Cell cycle stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a Cell Cycle Stress-Related Protein (CCSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated CCSRPs, and isolated nucleic acid coding CCSRPs, and vectors and host cells containing the latter.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S.Nonprovisional patent application Ser. No. 09/828,062 filed Apr. 6,2001, and claims the priority benefit of U.S. Provisional ApplicationSerial No. 60/196,001 filed Apr. 7, 2000, both of which are herebyincorporated in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to nucleic acid sequencesencoding proteins that are associated with abiotic stress responses andabiotic stress tolerance in plants. In particular, this inventionrelates to nucleic acid sequences encoding proteins that confer drought,cold, and/or salt tolerance to plants.

[0004] 2. Background Art

[0005] Abiotic environmental stresses, such as drought stress, salinitystress, heat stress, and cold stress, are major limiting factors ofplant growth and productivity. Crop losses and crop yield losses ofmajor crops such as rice, maize (corn) and wheat caused by thesestresses represent a significant economic and political factor andcontribute to food shortages in many underdeveloped countries.

[0006] Plants are typically exposed during their life cycle toconditions of reduced environmental water content. Most plants haveevolved strategies to protect themselves against these conditions ofdesiccation. However, if the severity and duration of the droughtconditions are too great, the effects on plant development, growth andyield of most crop plants are profound. Furthermore, most of the cropplants are very susceptible to higher salt concentrations in the soil.Continuous exposure to drought and high salt causes major alterations inthe plant metabolism. These great changes in metabolism ultimately leadto cell death and consequently yield losses.

[0007] Developing stress-tolerant plants is a strategy that has thepotential to solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold and salttolerance in model, drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely-unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

[0008] Therefore, what is needed is the identification of the genes andproteins involved in these multi-component processes leading to stresstolerance. Elucidating the function of genes expressed in stresstolerant plants will not only advance our understanding of plantadaptation and tolerance to environmental stresses, but also may provideimportant information for designing new strategies for crop improvement.

[0009] One group of proteins that seems to be associated with stressresponses in plants and animals are proteins related to cell divisionand the cell cycle. Evidence of this relationship is found in the factthat different environmental stresses related to soil water content,incident light and varying leaf temperature all result in a change inthe cell division rate. For example, in sunflower leaves, there is alengthening of the cell cycle associated with stress and aging. Cells inthe sunflower leaves tend to arrest at the G1 phase of cell cycle, withno change in the duration of the S-G2-M phases of the cell cycle.Similar observations have been made in other plant species placed underenvironmental stresses such as sucrose starvation (Van't Hof 1973Bookhaven Symp. 25:152), oxidative stress (Reichheld et al. 1999 PlantJ. 17:647), and water depletion (Schuppler et al. 1998 Plant Physiol.117:667). These observations and results suggest that there is animportant “checkpoint” in the regulation of the cell cycle at the G1-Stransition, while other studies suggest another “checkpoint” at the G2-Mtransition with an arrest of the cells at the G2 phase. It has also beendetermined that temperature affects the duration of all cell cyclephases by a similar proportion without the preferential arrest in anyparticular phase of the cell cycle (Tardieu and Granier, 2000 Plant Mol.Biol. 43:555). Although these aforementioned responses to environmentalstresses differ, each of these results demonstrates theinterconnectivity of the stress response system of these plants and thecell division proteins therein.

[0010] The prior art describes several proteins associated with celldivision and the cell cycle. One such protein, or protein complex, isthe cyclin-CDK complex, which controls progression of the cell cyclephases. Cyclin-dependent protein kinases (CDKs) require cyclin bindingfor activity and are now widely recognized players at the checkpoints ofthe eukaryotic cell cycle. The widespread importance of CDKs wasrealized nearly ten years ago from independent genetic approaches inyeast and biochemical studies of mitotic controls in fertilized eggs ofmarine invertebrates.

[0011] One known component of a CDK complex is a Cdc2 protein termed p₃₄^(cdc2), which is required at both control points (G1-S and G2-M).Several other Cdc2 homologs have been isolated from human and plantspecies including yeast. One such yeast homolog is Cdc48, which plays arole in the spindle pole body separation in Saccharomyces cerevisiae.Another Cdc2 homolog has been described in Arabidopsis (Feiler et al.1995 EMBO J 14:5626) that is highly expressed in the proliferating cellsof the vegetative shoot, root, floral inflorescence and flowers and inrapidly growing cells. The Arabidopsis Cdc48 gene is up regulated in thedeveloping microspores and ovules and down regulated in mostdifferentiated cell types. In addition, this gene has been localized tothe nucleus and during cytokinesis to the fragmoplast.

[0012] Another group of proteins involved in cell division and the cellcycle are pRB proteins. Growing evidence suggests that pRB-like proteinsin plants might be among the nuclear targets of plant CDKs. In mammals,the pRB is central to the regulation of the G1-to-S transition.Phosphorylation of mammalian pRB by cyclin D- and cyclin E-dependentkinases renders the pRB inactive and thereby represses the S phase andpromotes DNA replication. Significantly, the pRB-binding motif LXCXE(where X denotes any amino acid) is found in all known plant D cyclins.In plants, LXCXE-dependent interactions between D cyclins fromArabidopsis and maize pRB proteins have been demonstrated in vitro andin a yeast two-hybrid assay. The role of pRBs in plant cell divisionrelated signaling cascades is further supported by the fact that severalpRBs, and in particular, the maize pRB, contain multiple putative CDKphosphorylation sites and are efficiently phosphorylated in vitro bymammalian G1- and S-specific CDKs. Maize pRB proteins are also known toundergo changes in phosphorylation during the transition toendoreduplication in the endosperm. However, phosphorylation by plantCDKs remains to be demonstrated.

[0013] Therefore, although several plant cell cycle proteins have beenelucidated, the prior art has yet to describe the plant cell cyclesignal transduction cascades in detail. The prior art also fails todescribe the relation between plant cell cycle proteins and a plant'sresponse to environmental stress such that a stress tolerant plant canbe generated. Accordingly, there is a need to identify genes expressedin stress tolerant plants that have the capacity to confer stressresistance to its host plant and to other plant species. Newly generatedstress tolerant plants will have many advantages, such as increasing therange that crop plants can be cultivated by, for example, decreasing thewater requirements of a plant species.

SUMMARY OF THE INVENTION

[0014] This invention fulfills in part the need to identify new, uniquecell cycle proteins capable of conferring stress tolerance to plantsupon over-expression. The present invention provides a transgenic plantcell transformed by a Cell Cycle Stress-Related Protein (CCSRP) codingnucleic acid, wherein expression of the nucleic acid sequence in theplant cell results in increased tolerance to environmental stress ascompared to a wild type variety of the plant cell. Namely, describedherein are the cell cycle proteins 1) Cell Cycle-1 (CC-1); 2) CellCycle-2 (CC-2) and 3) Cell Cycle-3 (CC-3), all from Physcomitrellapatens.

[0015] The invention provides in some embodiments that the CCSRP andcoding nucleic acid are that found in members of the genusPhyscomitrella. In another preferred embodiment, the nucleic acid andprotein are from a Physcomitrella patens. The invention provides thatthe environmental stress can be salinity, drought, temperature, metal,chemical, pathogenic and oxidative stresses, or combinations thereof. Inpreferred embodiments, the environmental stress can be drought or coldtemperature.

[0016] The invention further provides a seed produced by a transgenicplant transformed by a CCSRP coding nucleic acid, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention furtherprovides a seed produced by a transgenic plant expressing a CCSRP,wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.

[0017] The invention further provides an agricultural product producedby any of the below-described transgenic plants, plant parts or seeds.The invention further provides an isolated CCSRP as described below. Theinvention further provides an isolated CCSRP coding nucleic acid,wherein the CCSRP coding nucleic acid codes for a CCSRP as describedbelow.

[0018] The invention further provides an isolated recombinant expressionvector comprising a CCSRP coding nucleic acid as described below,wherein expression of the vector in a host cell results in increasedtolerance to environmental stress as compared to a wild type variety ofthe host cell. The invention further provides a host cell containing thevector and a plant containing the host cell.

[0019] The invention further provides a method of producing a transgenicplant with a CCSRP coding nucleic acid, wherein expression of thenucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a CCSRP coding nucleic acid, and (b) generating from theplant cell a transgenic plant with an increased tolerance toenvironmental stress as compared to a wild type variety of the plant. Inpreferred embodiments, the CCSRP and CCSRP coding nucleic acid are asdescribed below.

[0020] The present invention further provides a method of identifying anovel CCSRP, comprising (a) raising a specific antibody response to aCCSRP, or fragment thereof, as described below; (b) screening putativeCCSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelCCSRP; and (c) identifying from the bound material a novel CCSRP incomparison to known CCSRP. Alternatively, hybridization with nucleicacid probes as described below can be used to identify novel CCSRPnucleic acids.

[0021] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a CCSRP inthe plant, wherein the CCSRP is as described below. The inventionprovides that this method can be performed such that the stresstolerance is either increased or decreased. Preferably, stress toleranceis increased in a plant via increasing expression of a CCSRP.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows a diagram of the plant expression vector ppBPSsc022containing the super promoter driving the expression of SEQ ID NO: 4, 5,or 6 (“Desired Gene”). The components are: NPTII kanamycin resistancegene, AtAct2-i promoter, OCS3 terminator, NOSpA terminator (Jefferson etal., 1987 EMBO J 6:3901-7).

[0023]FIG. 2 shows the results of a drought stress test withover-expressing PpCC-1 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0024]FIG. 3 shows the results of a freezing stress test withover-expressing PpCC-1 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0025]FIG. 4 shows the results of a drought stress test withover-expressing PpCC-2 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0026]FIG. 5 shows the results of a drought stress test withover-expressing PpCC-3 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. However, before thepresent compounds, compositions, and methods are disclosed anddescribed, it is to be understood that this invention is not limited tospecific nucleic acids, specific polypeptides, specific cell types,specific host cells, specific conditions, or specific methods, etc., assuch may, of course, vary, and the numerous modifications and variationstherein will be apparent to those skilled in the art. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing specific embodiments only and is not intended to be limiting.In particular, the designation of the amino acid sequences as protein“Cell Cycle Stress-Related Proteins” (CCSRPs), in no way limits thefunctionality of those sequences.

[0028] The present invention provides a transgenic plant celltransformed by a CCSRP coding nucleic acid, wherein expression of thenucleic acid sequence in the plant cell results in increased toleranceto environmental stress as compared to a wild type variety of the plantcell. The invention further provides transgenic plant parts andtransgenic plants containing the plant cells described herein. Alsoprovided is a plant seed produced by a transgenic plant transformed by aCCSRP coding nucleic acid, wherein the seed contains the CCSRP codingnucleic acid, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant. The invention further provides a seed produced by atransgenic plant expressing a CCSRP, wherein the seed contains theCCSRP, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention also provides an agricultural product produced by any ofthe below-described transgenic plants, plant parts and plant seeds.

[0029] As used herein, the term “variety” refers to a group of plantswithin a species that share constant characters that separate them fromthe typical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

[0030] The present invention describes for the first time that thePhyscomitrella patens CCSRPs, CC-1, CC-2 and CC-3, are useful forincreasing a plant's tolerance to environmental stress. Accordingly, thepresent invention provides isolated CCSRPs selected from the groupconsisting of CC-1, CC-2 and CC-3, and homologs thereof. In preferredembodiments, the CCSRP is selected from 1) a Cell Cycle-1 (CC-1) proteinas defined in SEQ ID NO:7; 2) a Cell Cycle-2 (CC-2) protein as definedin SEQ ID NO:8; and 3) a Cell Cycle-3 (CC-3) protein as defined in SEQID NO:9 and homologs and orthologs thereof. Homologs and orthologs ofthe amino acid sequences are defined below.

[0031] The CCSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedbelow), the expression vector is introduced into a host cell (asdescribed below) and the CCSRP is expressed in the host cell. The CCSRPcan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a CCSRP polypeptide, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native CCSRP can be isolated from cells (e.g., Physcomitrellapatens), for example using an anti-CCSRP antibody, which can be producedby standard techniques utilizing a CCSRP or fragment thereof.

[0032] The invention further provides an isolated CCSRP coding nucleicacid. The present invention includes CCSRP coding nucleic acids thatencode CCSRPs as described herein. In preferred embodiments, the CCSRPcoding nucleic acid is selected from 1) a Cell Cycle-1 (CC-1) nucleicacid as defined in SEQ ID NO:4; 2) a Cell Cycle-2 (CC-2) nucleic acid asdefined in SEQ ID NO:5; and 3) a Cell Cycle-3 (CC-3) nucleic acid asdefined in SEQ ID NO:6 and homologs and orthologs thereof. Homologs andorthologs of the nucleotide sequences are defined below. In onepreferred embodiment, the nucleic acid and protein are isolated from theplant genus Physcomitrella. In another preferred embodiment, the nucleicacid and protein are from a Physcomitrella patens (P. patens) plant.

[0033] As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, or temperature, or combinations thereof, and inparticular, can be high salinity, low water content or low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

[0034] As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0035] An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of some of the sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated CCSRP nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived (e.g., a Physcomitrella patens cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

[0036] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a P. patens CCSRP cDNA can be isolated from a P.patens library using all or portion of one of the sequences of SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3. Moreover, a nucleic acid moleculeencompassing all or a portion of one of the sequences of SEQ ID NO:1,SEQ ID NO:2 and SEQ ID NO:3 can be isolated by the polymerase chainreaction using oligonucleotide primers designed based upon thissequence. For example, mRNA can be isolated from plant cells (e.g., bythe guanidinium-thiocyanate extraction procedure of Chirgwin et al.,1979 Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3. A nucleic acid molecule of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a CCSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

[0037] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises one of the nucleotide sequences shown in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6. These cDNAs comprise sequencesencoding the CCSRPs (i.e., the “coding region”, indicated in Table 1),as well as 5′ untranslated sequences and 3′ untranslated sequences. Itis to be understood that SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6comprise both coding regions and 5′ and 3′ untranslated regions.Alternatively, the nucleic acid molecules of the present invention cancomprise only the coding region of any of the sequences in SEQ ID NO:4,SEQ ID NO:5 or SEQ ID NO:6 or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as an “ORF position”. The present invention also includesCCSRP coding nucleic acids that encode CCSRPs as described herein.Preferred is a CCSRP coding nucleic acid that encodes a CCSRP selectedfrom the group consisting of, CC-1 (SEQ ID NO:7), CC-2 (SEQ ID NO:8) andCC-3 (SEQ ID NO:9).

[0038] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the coding region of one of the sequences in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, for example, a fragment which can beused as a probe or primer or a fragment encoding a biologically activeportion of a CCSRP. The nucleotide sequences determined from the cloningof the CCSRP genes from P. patens allow for the generation of probes andprimers designed for use in identifying and/or cloning CCSRP homologs inother cell types and organisms, as well as CCSRP homologs from othermosses and related species.

[0039] Portions of proteins encoded by the CCSRP nucleic acid moleculesof the invention are preferably biologically active portions of one ofthe CCSRPs described herein. As used herein, the term “biologicallyactive portion of” a CCSRP is intended to include a portion, e.g., adomain/motif, of a CCSRP that participates in a stress toleranceresponse in a plant, has an activity as set forth in Table 1, orparticipates in the transcription of a protein involved in a stresstolerance response in a plant. To determine whether a CCSRP, or abiologically active portion thereof, can participate in transcription ofa protein involved in a stress tolerance response in a plant, a stressanalysis of a plant comprising the CCSRP may be performed. Such analysismethods are well known to those skilled in the art, as detailed inExample 7. More specifically, nucleic acid fragments encodingbiologically active portions of a CCSRP can be prepared by isolating aportion of one of the sequences in SEQ ID NO:7, SEQ ID NO:8 and SEQ IDNO:9, expressing the encoded portion of the CCSRP or peptide (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the CCSRP or peptide.

[0040] Biologically active portions of a CCSRP are encompassed by thepresent invention and include peptides comprising amino acid sequencesderived from the amino acid sequence of a CCSRP, e.g., an amino acidsequence of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, or the amino acidsequence of a protein homologous to a CCSRP, which include fewer aminoacids than a full length CCSRP or the full length protein which ishomologous to a CCSRP, and exhibit at least one activity of a CCSRP.Typically, biologically active portions (e.g., peptides which are, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif with at least oneactivity of a CCSRP. Moreover, other biologically active portions inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the activitiesdescribed herein. Preferably, the biologically active portions of aCCSRP include one or more selected domains/motifs or portions thereofhaving biological activity.

[0041] The invention also provides CCSRP chimeric or fusion proteins. Asused herein, a CCSRP “chimeric protein” or “fusion protein” comprises aCCSRP polypeptide operatively linked to a non-CCSRP polypeptide. A CCSRPpolypeptide refers to a polypeptide having an amino acid sequencecorresponding to a CCSRP, whereas a non-CCSRP polypeptide refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the CCSRP, e.g., a protein thatis different from the CCSRP and is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the CCSRP polypeptide and the non-CCSRPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-CCSRPpolypeptide can be fused to the N-terminus or C-terminus of the CCSRPpolypeptide. For example, in one embodiment, the fusion protein is aGST-CCSRP fusion protein in which the CCSRP sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant CCSRPs. In another embodiment, the fusionprotein is a CCSRP containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a CCSRP can be increased through use of aheterologous signal sequence.

[0042] Preferably, a CCSRP chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A CCSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the CCSRP.

[0043] In addition to fragments and fusion proteins of the CCSRPsdescribed herein, the present invention includes homologs and analogs ofnaturally occurring CCSRPs and CCSRP encoding nucleic acids in a plant.“Homologs” are defined herein as two nucleic acids or proteins that havesimilar, or “homologous”, nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists and antagonists of CCSRPs as defined hereafter. The term“homolog” further encompasses nucleic acid molecules that differ fromone of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6 (and portions thereof) due to degeneracy of the genetic codeand thus encode the same CCSRP as that encoded by the nucleotidesequences shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. As usedherein a “naturally occurring” CCSRP refers to a CCSRP amino acidsequence that occurs in nature. Preferably, a naturally occurring CCSRPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0044] An agonist of the CCSRP can retain substantially the same, or asubset, of the biological activities of the CCSRP. An antagonist of theCCSRP can inhibit one or more of the activities of the naturallyoccurring form of the CCSRP. For example, the CCSRP antagonist cancompetitively bind to a downstream or upstream member of the cellmembrane component metabolic cascade that includes the CCSRP, or bind toa CCSRP that mediates transport of compounds across such membranes,thereby preventing translocation from taking place.

[0045] Nucleic acid molecules corresponding to natural allelic variantsand analogs, orthologs and paralogs of a CCSRP cDNA can be isolatedbased on their identity to the Physcomitrella patens CCSRP nucleic acidsdescribed herein using CCSRP cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. In an alternative embodiment,homologs of the CCSRP can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the CCSRP for CCSRPagonist or antagonist activity. In one embodiment, a variegated libraryof CCSRP variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of CCSRP variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential CCSRP sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofCCSRP sequences therein. There are a variety of methods that can be usedto produce libraries of potential CCSRP homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene is then ligated into an appropriate expression vector.Use of a degenerate set of genes allows for the provision, in onemixture, of all of the sequences encoding the desired set of potentialCCSRP sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A., 1983 Tetrahedron 39:3;Itakura et al., 1984 Annu. Rev. Biochem. 53:323; Itakura et al., 1984Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

[0046] In addition, libraries of fragments of the CCSRP coding regionscan be used to generate a variegated population of CCSRP fragments forscreening and subsequent selection of homologs of a CCSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a CCSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with SI nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the CCSRP.

[0047] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of CCSRPhomologs. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique that enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify CCSRP homologs (Arkin and Yourvan, 1992PNAS 89:7811-7815; Delgrave et al., 1993 Protein Engineering6(3):327-331). In another embodiment, cell based assays can be exploitedto analyze a variegated CCSRP library, using methods well known in theart. The present invention further provides a method of identifying anovel CCSRP, comprising (a) raising a specific antibody response to aCCSRP, or a fragment thereof, as described above; (b) screening putativeCCSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelCCSRP; and (c) analyzing the bound material in comparison to knownCCSRP, to determine its novelty.

[0048] To determine the percent homology of two amino acid sequences(e.g., one of the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9and a mutant form thereof), the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of oneprotein or nucleic acid for optimal alignment with the other protein ornucleic acid). The amino acid residues at corresponding amino acidpositions or nucleotide positions are then compared. When a position inone sequence (e.g., one of the sequences of SEQ ID NO:7, SEQ ID NO:8 andSEQ ID NO:9) is occupied by the same amino acid residue as thecorresponding position in the other sequence (e.g., a mutant form of thesequence selected from the polypeptide of SEQ ID NO:7, SEQ ID NO:8 andSEQ ID NO:9), then the molecules are homologous at that position (i.e.,as used herein amino acid or nucleic acid “homology” is equivalent toamino acid or nucleic acid “identity”). The same type of comparison canbe made between two nucleic acid sequences.

[0049] The percent homology between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., %homology=numbers of identical positions/total numbers of positions×100).Preferably, the amino acid sequences included in the present inventionare at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80%, 80-90%, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In yetanother embodiment, at least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence encoded by a nucleic acid sequence shownin SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. In other embodiments, thepreferable length of sequence comparison for proteins is at least 15amino acid residues, more preferably at least 25 amino acid residues,and most preferably at least 35 amino acid residues.

[0050] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence which is atleast about 50-60%, preferably at least about 60-70%, more preferably atleast about 70-80%, 80-90%, or 90-95%, and even more preferably at leastabout 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotidesequence shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a portionthereof. The preferable length of sequence comparison for nucleic acidsis at least 75 nucleotides, more preferably at least 100 nucleotides andmost preferably the entire coding region of the nucleic acid.

[0051] It is also preferable that the homologous nucleic acid moleculeof the invention encodes a protein or portion thereof which includes anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 such that theprotein or portion thereof maintains the same or a similar function asthe amino acid sequence to which it is compared. Functions of the CCSRPamino acid sequences of the present invention include the ability toparticipate in a stress tolerance response in a plant, or moreparticularly, to participate in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant.Examples of such activities are described in Table 1.

[0052] In addition to the above-described methods, a determination ofthe percent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403-410).

[0053] BLAST nucleic acid searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleic acid sequenceshomologous to the CCSRP nucleic acid molecules of the invention.Additionally, BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to CCSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used to obtain amino acid sequenceshomologous to the CCSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used.

[0054] Finally, homology between nucleic acid sequences can also bedetermined using hybridization techniques known to those of skill in theart. Accordingly, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to one of the nucleotide sequences shown in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, or a portion thereof. Moreparticularly, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:4, SEQ ID NO:5 or SEQ ID NO:6. In other embodiments, the nucleic acidis at least 30, 50, 100, 250 or more nucleotides in length.

[0055] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, 6.3.1-6.3.6, John Wiley& Sons, N.Y. (1989). A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 corresponds to anaturally occurring nucleic acid molecule. As used herein, a “naturallyoccurring” nucleic acid molecule refers to an RNA or DNA molecule havinga nucleotide sequence that occurs in nature (e.g., encodes a naturalprotein). In one embodiment, the nucleic acid encodes a naturallyoccurring Physcomitrella patens CCSRP.

[0056] Using the above-described methods, and others known to those ofskill in the art, one of ordinary skill in the art can isolate homologsof the CCSRPs comprising amino acid sequences shown in SEQ ID NO:7, SEQID NO:8 or SEQ ID NO:9. One subset of these homologs is allelicvariants. As used herein, the term “allelic variant” refers to anucleotide sequence containing polymorphisms that lead to changes in theamino acid sequences of a CCSRP and that exist within a naturalpopulation (e.g., a plant species or variety). Such natural allelicvariations can typically result in 1-5% variance in a CCSRP nucleicacid. Allelic variants can be identified by sequencing the nucleic acidsequence of interest in a number of different plants, which can bereadily carried out by using hybridization probes to identify the sameCCSRP genetic locus in those plants. Any and all such nucleic acidvariations and resulting amino acid polymorphisms or variations in aCCSRP that are the result of natural allelic variation and that do notalter the functional activity of a CCSRP, are intended to be within thescope of the invention.

[0057] Moreover, nucleic acid molecules encoding CCSRPs from the same orother species such as CCSRP analogs, orthologs and paralogs, areintended to be within the scope of the present invention. As usedherein, the term “analogs” refers to two nucleic acids that have thesame or similar function, but that have evolved separately in unrelatedorganisms. As used herein, the term “orthologs” refers to two nucleicacids from different species, but that have evolved from a commonancestral gene by speciation. Normally, orthologs encode proteins havingthe same or similar functions. As also used herein, the term “paralogs”refers to two nucleic acids that are related by duplication within agenome. Paralogs usually have different functions, but these functionsmay be related (Tatusov, R. L. et al. 1997 Science 278(5338):631-637).Analogs, orthologs and paralogs of a naturally occurring CCSRP candiffer from the naturally occurring CCSRP by post-translationalmodifications, by amino acid sequence differences, or by both.Post-translational modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation, and such modifications may occurduring polypeptide synthesis or processing or following treatment withisolated modifying enzymes. In particular, orthologs of the inventionwill generally exhibit at least 80-85%, more preferably 90%, and mostpreferably 95%, 96%, 97%, 98% or even 99% identity or homology with allor part of a naturally occurring CCSRP amino acid sequence and willexhibit a function similar to a CCSRP. Orthologs of the presentinvention are also preferably capable of participating in the stressresponse in plants. In one embodiment, the CCSRP orthologs maintain theability to participate in the metabolism of compounds necessary for theconstruction of cellular membranes in Physcomitrella patens, or in thetransport of molecules across these membranes.

[0058] In addition to naturally-occurring variants of a CCSRP sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, thereby leading tochanges in the amino acid sequence of the encoded CCSRP, withoutaltering the functional ability of the CCSRP. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in a sequence of SEQ ID NO:4, SEQ IDNO:5 or SEQ ID NO:6. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of one of the CCSRPswithout altering the activity of said CCSRP, whereas an “essential”amino acid residue is required for CCSRP activity. Other amino acidresidues, however, (e.g., those that are not conserved or onlysemi-conserved in the domain having CCSRP activity) may not be essentialfor activity and thus are likely to be amenable to alteration withoutaltering CCSRP activity.

[0059] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding CCSRPs that contain changes in amino acidresidues that are not essential for CCSRP activity. Such CCSRPs differin amino acid sequence from a sequence contained in SEQ ID NO:7, SEQ IDNO:8 or SEQ ID NO:9, yet retain at least one of the CCSRP activitiesdescribed herein. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50% homologous to anamino acid sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 50-60% homologous to one of the sequences of SEQ ID NO:7, SEQ IDNO:8 and SEQ ID NO:9, more preferably at least about 60-70% homologousto one of the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,even more preferably at least about 70-80%, 80-90%, 90-95% homologous toone of the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, andmost preferably at least about 96%, 97%, 98%, or 99% homologous to oneof the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. Thepreferred CCSRP homologs of the present invention are preferably capableof participating in the a stress tolerance response in a plant, or moreparticularly, participating in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant, or haveone or more activities set forth in Table 1.

[0060] An isolated nucleic acid molecule encoding a CCSRP homologous toa protein sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:6 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain.

[0061] Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a CCSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a CCSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a CCSRP activity described herein to identify mutants that retainCCSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined byanalyzing the stress tolerance of a plant expressing the protein asdescribed in Example 7.

[0062] In addition to the nucleic acid molecules encoding the CCSRPsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules that are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence that is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire CCSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a CCSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of , , , comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding a CCSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0063] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6, or a portion thereof. A nucleic acid molecule that iscomplementary to one of the nucleotide sequences shown in SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6 is one which is sufficiently complementaryto one of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6 such that it can hybridize to one of the nucleotidesequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, therebyforming a stable duplex.

[0064] Given the coding strand sequences encoding the CCSRPs disclosedherein (e.g., the sequences set forth in SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof CCSRP mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of CCSRPmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of CCSRP mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length.

[0065] An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0066] The antisense nucleic acid molecules of the invention aretypically administered to a cell or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aCCSRP to thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

[0067] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al., 1987 Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

[0068] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes described inHaselhoff and Gerlach, 1988 Nature 334:585-591) can be used tocatalytically cleave CCSRP mRNA transcripts to thereby inhibittranslation of CCSRP mRNA. A ribozyme having specificity for aCCSRP-encoding nucleic acid can be designed based upon the nucleotidesequence of a CCSRP cDNA, as disclosed herein (i.e., SEQ ID NO:4, SEQ IDNO:5 or SEQ ID NO:6) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a CCSRP-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, CCSRP mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W., 1993 Science261:1411-1418.

[0069] Alternatively, CCSRP gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofa CCSRP nucleotide sequence (e.g., a CCSRP promoter and/or enhancer) toform triple helical structures that prevent transcription of a CCSRPgene in target cells. See generally, Helene, C., 1991 Anticancer DrugDes. 6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J., 1992 Bioassays 14(12):807-15.

[0070] In addition to the CCSRP nucleic acids and proteins describedabove, the present invention encompasses these nucleic acids andproteins attached to a moiety. These moieties include, but are notlimited to, detection moieties, hybridization moieties, purificationmoieties, delivery moieties, reaction moieties, binding moieties, andthe like. A typical group of nucleic acids attached to a moiety areprobes and primers. Probes and primers typically comprise asubstantially isolated oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably about 25, morepreferably about 40, 50 or 75 consecutive nucleotides of a sense strandof one of the sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:6, an anti-sense sequence of one of the sequences set forth in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, or naturally occurring mutantsthereof. Primers based on a nucleotide sequence of SEQ ID NO:4, SEQ IDNO:5 or SEQ ID NO:6 can be used in PCR reactions to clone CCSRPhomologs. Probes based on the CCSRP nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a genomic marker test kit for identifying cellswhich express a CCSRP, such as by measuring a level of a CCSRP-encodingnucleic acid, in a sample of cells, e.g., detecting CCSRP mRNA levels ordetermining whether a genomic CCSRP gene has been mutated or deleted.

[0071] In particular, a useful method to ascertain the level oftranscription of the gene (an indicator of the amount of mRNA availablefor translation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York). This information at least partiallydemonstrates the degree of transcription of the transformed gene. Totalcellular RNA can be prepared from cells, tissues or organs by severalmethods, all well-known in the art, such as that described in Bormann,E. R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence orrelative quantity of protein translated from this mRNA, standardtechniques, such as a Western blot, may be employed. These techniquesare well known to one of ordinary skill in the art. (See, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork).

[0072] The invention further provides an isolated recombinant expressionvector comprising a CCSRP nucleic acid as described above, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0073] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,89-108, CRC Press: Boca Raton, Fla., including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., CCSRPs, mutant forms of CCSRPs, fusionproteins, etc.).

[0074] The recombinant expression vectors of the invention can bedesigned for expression of CCSRPs in prokaryotic or eukaryotic cells.For example, CCSRP genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al., 1992 Foreign geneexpression in yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M.J. J. et al., 1991 Heterologous gene expression in filamentous fungi,in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds.,p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J.J. & Punt, P. J., 1991 Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al., 1999 Marine Biotechnology 1(3):239-251), ciliates ofthe types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in PCT Application No. WO 98/01572 and multicellularplant cells (see Schmidt, R. and Willmitzer, L., 1988 High efficiencyAgrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana leaf and cotyledon explants, Plant Cell Rep. 583-586); PlantMolecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter6/7, S.71-119 (1993); F. F. White, B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,eds. Kung und R. Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu.Rev. Plant Physiol. Plant Molec. Biol. 42:205-225 and references citedtherein) or mammalian cells. Suitable host cells are discussed furtherin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press: San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0075] Expression of proteins in prokaryotes is most often carried outwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, usually to theamino terminus of the recombinant protein but also to the C-terminus orfused within suitable regions in the proteins. Such fusion vectorstypically serve three purposes: 1) to increase expression of arecombinant protein; 2) to increase the solubility of a recombinantprotein; and 3) to aid in the purification of a recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.

[0076] Typical fusion expression vectors include pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the CCSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant CCSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

[0077] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a co-expressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0078] One strategy to maximize recombinant protein expression is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

[0079] In another embodiment, the CCSRP expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., 1987 EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz etal., 1987 Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Vectors and methods for the construction of vectorsappropriate for use in other fungi, such as the filamentous fungi,include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J.(1991) “Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al.,eds., p. 1-28, Cambridge University Press: Cambridge.

[0080] Alternatively, the CCSRPs of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

[0081] In yet another embodiment, a CCSRP nucleic acid of the inventionis expressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2^(nd,) ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

[0082] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., 1987 Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton, 1988 Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989 EMBO J. 8:729-733) and immunoglobulins (Baneiji et al.,1983 Cell 33:729-740; Queen and Baltimore, 1983 Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle, 1989 PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal., 1985 Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally regulatedpromoters are also encompassed, for example, the murine hox promoters(Kessel and Gruss, 1990 Science 249:374-379) and the fetoproteinpromoter (Campes and Tilghman, 1989 Genes Dev. 3:537-546).

[0083] In another embodiment, the CCSRPs of the invention may beexpressed in unicellular plant cells (such as algae) (see Falciatore etal., 1999 Marine Biotechnology 1(3):239-251 and references therein) andplant cells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

[0084] A plant expression cassette preferably contains regulatorysequences capable of driving gene expression in plant cells and operablylinked so that each sequence can fulfill its function, for example,termination of transcription by polyadenylation signals. Preferredpolyadenylation signals are those originating from Agrobacteriumtumefaciens t-DNA such as the gene 3 known as octopine synthase of theTi-plasmid pTiACH5 (Gielen et al., 1984 EMBO J. 3:835) or functionalequivalents thereof but also all other terminators functionally activein plants are suitable.

[0085] As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

[0086] Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

[0087] Other preferred sequences for use in plant gene expressioncassettes are targeting-sequences necessary to direct the gene productin its appropriate cell compartment (for review see Kermode, 1996 Crit.Rev. Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

[0088] Plant gene expression can also be facilitated via an induciblepromoter (for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol.Biol. 48:89-108). Chemically inducible promoters are especially suitableif gene expression is wanted to occur in a time specific manner.Examples of such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992 Plant J. 2:397-404) and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

[0089] Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814) orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al. (1993 Mol. Gen.Genet. 236:331-340).

[0090] Especially preferred are those promoters that confer geneexpression in specific tissues and organs, such as guard cells and theroot hair cells. Suitable promoters include the napin-gene promoter fromrapeseed (U.S. Pat. No. 5,608,152), the U.S. Pat. No.-promoter fromVicia faba (Baeumlein et al., 1991 Mol Gen Genet. 225(3):459-67), theoleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), thephaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200),the Bce4-promoter from Brassica (PCT Application No. WO 91/13980) or thelegumin B4 promoter (LeB4; Baeumlein et al., 1992 Plant Journal,2(2):233-9) as well as promoters conferring seed specific expression inmonocot plants like maize, barley, wheat, rye, rice, etc. Suitablepromoters to note are the 1pt2 or 1pt1-gene promoter from barley (PCTApplication No. WO 95/15389 and PCT Application No. WO 95/23230) orthose described in PCT Application No. WO 99/16890 (promoters from thebarley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamingene, wheat gliadin gene, wheat glutelin gene, maize zein gene, oatglutelin gene, Sorghum kasirin-gene and rye secalin gene).

[0091] Also especially suited are promoters that confer plastid-specificgene expression since plastids are the compartment where lipidbiosynthesis occurs. Suitable promoters are the viral RNA-polymerasepromoter described in PCT Application No. WO 95/16783 and PCTApplication No. WO 97/06250 and the clpP-promoter from Arabidopsisdescribed in PCT Application No. WO 99/46394.

[0092] The invention further provides a recombinant expression vectorcomprising a CCSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a CCSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986 and Mol et al.,1990 FEBS Letters 268:427-430.

[0093] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but they also apply to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0094] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a CCSRP can be expressed in bacterial cells such as C.glutamicum, insect cells, fungal cells or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

[0095] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.2^(nd,) ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratorymanuals such as Methods in Molecular Biology, 1995, Vol. 44,Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa,New Jersey. As biotic and abiotic stress tolerance is a general traitwished to be inherited into a wide variety of plants like maize, wheat,rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed andcanola, manihot, pepper, sunflower and tagetes, solanaceous plants likepotato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa,bushy plants (coffee, cacao, tea), Salix species, trees (oil palm,coconut), perennial grasses and forage crops, these crop plants are alsopreferred target plants for a genetic engineering as one furtherembodiment of the present invention.

[0096] In particular, the invention provides a method of producing atransgenic plant with a CCSRP coding nucleic acid, wherein expression ofthe nucleic acid(s) in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a CCSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention alsoprovides a method of increasing expression of a gene of interest withina host cell as compared to a wild type variety of the host cell, whereinthe gene of interest is transcribed in response to a CCSRP, comprising:(a) transforming the host cell with an expression vector comprising aCCSRP coding nucleic acid, and (b) expressing the CCSRP within the hostcell, thereby increasing the expression of the gene transcribed inresponse to the CCSRP, as compared to a wild type variety of the hostcell.

[0097] For such plant transformation, binary vectors such as pBinAR canbe used (Höfgen and Willmitzer, 1990 Plant Science 66:221-230).Construction of the binary vectors can be performed by ligation of thecDNA in sense or antisense orientation into the T-DNA. 5-prime to thecDNA a plant promoter activates transcription of the cDNA. Apolyadenylation sequence is located 3-prime to the cDNA. Tissue-specificexpression can be achieved by using a tissue specific promoter. Forexample, seed-specific expression can be achieved by cloning the napinor LeB4 or USP promoter 5-prime to the cDNA. Also, any other seedspecific promoter element can be used. For constitutive expressionwithin the whole plant, the CaMV 35S promoter can be used. The expressedprotein can be targeted to a cellular compartment using a signalpeptide, for example for plastids, mitochondria or endoplasmic reticulum(Kermode, 1996 Crit. Rev. Plant Sci. 4 (15):285-423). The signal peptideis cloned 5-prime in frame to the cDNA to archive subcellularlocalization of the fusion protein. Additionally, promoters that areresponsive to abiotic stresses can be used with, such as the Arabidopsispromoter RD29A, the nucleic acid sequences disclosed herein. One skilledin the art will recognize that the promoter used should be operativelylinked to the nucleic acid such that the promoter causes transcriptionof the nucleic acid which results in the synthesis of a mRNA whichencodes a polypeptide. Alternatively, the RNA can be an antisense RNAfor use in affecting subsequent expression of the same or another geneor genes.

[0098] Alternate methods of transfection include the direct transfer ofDNA into developing flowers via electroporation or Agrobacteriummediated gene transfer. Agrobacterium mediated plant transformation canbe performed using for example the GV3101(pMP90) (Koncz and Schell, 1986Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain. Transformation can be performed by standardtransformation and regeneration techniques (Deblaere et al., 1994 Nucl.Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A,Plant Molecular Biology Manual, 2^(nd) Ed.—Dordrecht: Kluwer AcademicPubl., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in PlantMolecular Biology and Biotechnology, Boca Raton : CRC Press, 1993 360S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed viacotyledon or hypocotyl transformation (Moloney et al., 1989 Plant cellReport 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Useof antibiotica for Agrobacterium and plant selection depends on thebinary vector and the Agrobacterium strain used for transformation.Rapeseed selection is normally performed using kanamycin as selectableplant marker. Agrobacterium mediated gene transfer to flax can beperformed using, for example, a technique described by Mlynarova et al.,1994 Plant Cell Report 13:282-285. Additionally, transformation ofsoybean can be performed using for example a technique described inEuropean Patent No. 0424 047, U.S. Pat. No. 5,322,783, European PatentNo. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770.Transformation of maize can be achieved by particle bombardment,polyethylene glycol mediated DNA uptake or via the silicon carbide fibertechnique. (See, for example, Freeling and Walbot “The maize handbook”Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific exampleof maize transformation is found in U.S. Pat. No. 5,990,387 and aspecific example of wheat transformation can be found in PCT ApplicationNo. WO 93/07256.

[0099] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate or inplants that confer resistance towards a herbicide such as glyphosate orglufosinate. Nucleic acid molecules encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding a CCSRPor can be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid molecule can be identified by, for example,drug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

[0100] To create a homologous recombinant microorganism, a vector isprepared which contains at least a portion of a CCSRP gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the CCSRP gene. Preferably, the CCSRP geneis a Physcomitrella patens CCSRP gene, but it can be a homolog from arelated plant or even from a mammalian, yeast, or insect source. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous CCSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous CCSRP gene is mutated orotherwise altered but still encodes a functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous CCSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

[0101] Whereas in the homologous recombination vector, the alteredportion of the CCSRP gene is flanked at its 5′ and 3′ ends by anadditional nucleic acid molecule of the CCSRP gene to allow forhomologous recombination to occur between the exogenous CCSRP genecarried by the vector and an endogenous CCSRP gene, in a microorganismor plant. The additional flanking CCSRP nucleic acid molecule is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several hundreds of base pairs up tokilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R., and Capecchi, M. R., 1987 Cell51:503 for a description of homologous recombination vectors or Streppet al., 1998 PNAS, 95 (8):4368-4373 for cDNA based recombination inPhyscomitrella patens). The vector is introduced into a microorganism orplant cell (e.g., via polyethylene glycol mediated DNA), and cells inwhich the introduced CCSRP gene has homologously recombined with theendogenous CCSRP gene are selected using art-known techniques.

[0102] In another embodiment, recombinant microorganisms can be producedthat contain selected systems which allow for regulated expression ofthe introduced gene. For example, inclusion of a CCSRP gene on a vectorplacing it under control of the lac operon permits expression of theCCSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

[0103] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a CCSRP.Accordingly, the invention further provides methods for producing CCSRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a CCSRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered CCSRP) in a suitable medium until CCSRP is produced. In another embodiment,the method further comprises isolating CCSRPs from the medium or thehost cell.

[0104] Another aspect of the invention pertains to isolated CCSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof CCSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a CCSRP having less thanabout 30% (by dry weight) of non-CCSRP material (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-CCSRP material, still more preferably less than about 10% ofnon-CCSRP material, and most preferably less than about 5% non-CCSRPmaterial.

[0105] When the CCSRP or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofCCSRP in which the protein is separated from chemical precursors orother chemicals that are involved in the synthesis of the protein. Inone embodiment, the language “substantially free of chemical precursorsor other chemicals” includes preparations of a CCSRP having less thanabout 30% (by dry weight) of chemical precursors or non-CCSRP chemicals,more preferably less than about 20% chemical precursors or non-CCSRPchemicals, still more preferably less than about 10% chemical precursorsor non-CCSRP chemicals, and most preferably less than about 5% chemicalprecursors or non-CCSRP chemicals. In preferred embodiments, isolatedproteins, or biologically active portions thereof, lack contaminatingproteins from the same organism from which the CCSRP is derived.Typically, such proteins are produced by recombinant expression of, forexample, a Physcomitrella patens CCSRP in plants other thanPhyscomitrella patens or microorganisms such as C. glutamicum, ciliates,algae or fungi.

[0106] The nucleic acid molecules, proteins, protein homologs, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification ofPhyscomitrella patens and related organisms; mapping of genomes oforganisms related to Physcomitrella patens; identification andlocalization of Physcomitrella patens sequences of interest;evolutionary studies; determination of CCSRP regions required forfunction; modulation of a CCSRP activity; modulation of the metabolismof one or more cell functions; modulation of the transmembrane transportof one or more compounds; and modulation of stress resistance.

[0107] The moss Physcomitrella patens represents one member of themosses. It is related to other mosses such as Ceratodon purpureus whichis capable of growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

[0108] The CCSRP nucleic acid molecules of the invention have a varietyof uses. Most importantly, the nucleic acid and amino acid sequences ofthe present invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aCCSRP nucleic acid, wherein expression of the nucleic acid sequence inthe plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant. The transgenic plant canbe a monocot or a dicot. The invention further provides that thetransgenic plant can be selected from maize, wheat, rye, oat, triticale,rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot,pepper, sunflower, tagetes, solanaceous plants, potato, tobacco,eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salixspecies, oil palm, coconut, perennial grass and forage crops, forexample.

[0109] In particular, the present invention describes using theexpression of CC-1, CC-2 and CC-3 of Physcomitrella patens to engineerdrought-tolerant, salt-tolerant and/or cold-tolerant plants. Thisstrategy has herein been demonstrated for Arabidopsis thaliana,Rapeseed/Canola, soybeans, corn and wheat but its application is notrestricted to these plants. Accordingly, the invention provides atransgenic plant containing a CCSRP selected from CC-1 (SEQ ID NO:7),CC-2 (SEQ ID NO:8) and CC-3 (SEQ ID NO:9), wherein the environmentalstress is drought, increased salt or decreased or increased temperature.In preferred embodiments, the environmental stress is drought ordecreased temperature.

[0110] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a CCSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased. Inparticular, the present invention provides methods of producing atransgenic plant having an increased tolerance to environmental stressas compared to a wild type variety of the plant comprising increasingexpression of a CCSRP in a plant.

[0111] The methods of increasing expression of CCSRPs can be usedwherein the plant is either transgenic or not transgenic. In cases whenthe plant is transgenic, the plant can be transformed with a vectorcontaining any of the above described CCSRP coding nucleic acids, or theplant can be transformed with a promoter that directs expression ofnative CCSRP in the plant, for example. The invention provides that sucha promoter can be tissue specific. Furthermore, such a promoter can bedevelopmentally regulated. Alternatively, non-transgenic plants can havenative CCSRP expression modified by inducing a native promoter.

[0112] The expression of CC-1 (SEQ ID NO:7), CC-2 (SEQ ID NO:8) or CC-3(SEQ ID NO:9) in target plants can be accomplished by, but is notlimited to, one of the following examples: (a) constitutive promoter,(b) stress-inducible promoter, (c) chemical-induced promoter, and (d)engineered promoter over-expression with for example zinc-finger derivedtranscription factors (Greisman and Pabo, 1997 Science 275:657). Thelater case involves identification of the CC-1 (SEQ ID NO:7), CC-2 (SEQID NO:8) or CC-3 (SEQ ID NO:9) homologs in the target plant as well asfrom its promoter. Zinc-finger-containing recombinant transcriptionfactors are engineered to specifically interact with the CC-1 (SEQ IDNO:7), CC-2 (SEQ ID NO:8) or CC-3 (SEQ ID NO:9) homolog andtranscription of the corresponding gene is activated.

[0113] In addition to introducing the CCSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens or a close relative thereof.Also, they may be used to identify the presence of Physcomitrella patensor a relative thereof in a mixed population of microorganisms. Theinvention provides the nucleic acid sequences of a number ofPhyscomitrella patens genes; by probing the extracted genomic DNA of aculture of a unique or mixed population of microorganisms understringent conditions with a probe spanning a region of a Physcomitrellapatens gene which is unique to this organism, one can ascertain whetherthis organism is present.

[0114] Further, the nucleic acid and protein molecules of the inventionmay serve as markers for specific regions of the genome. This hasutility not only in the mapping of the genome, but also in functionalstudies of Physcomitrella patens proteins. For example, to identify theregion of the genome to which a particular Physcomitrella patensDNA-binding protein binds, the Physcomitrella patens genome could bedigested, and the fragments incubated with the DNA-binding protein.Those fragments that bind the protein may be additionally probed withthe nucleic acid molecules of the invention, preferably with readilydetectable labels. Binding of such a nucleic acid molecule to the genomefragment enables the localization of the fragment to the genome map ofPhyscomitrella patens, and, when performed multiple times with differentenzymes, facilitates a rapid determination of the nucleic acid sequenceto which the protein binds. Further, the nucleic acid molecules of theinvention may be sufficiently homologous to the sequences of relatedspecies such that these nucleic acid molecules may serve as markers forthe construction of a genomic map in related mosses.

[0115] The CCSRP nucleic acid molecules of the invention are also usefulfor evolutionary and protein structural studies. The metabolic andtransport processes in which the molecules of the invention participateare utilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein that are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

[0116] Manipulation of the CCSRP nucleic acid molecules of the inventionmay result in the production of CCSRPs having functional differencesfrom the wild-type CCSRPs. These proteins may be improved in efficiencyor activity, may be present in greater numbers in the cell than isusual, or may be decreased in efficiency or activity.

[0117] There are a number of mechanisms by which the alteration of aCCSRP of the invention may directly affect stress response and/or stresstolerance. In the case of plants expressing CCSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export ionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

[0118] The effect of the genetic modification in plants, C. glutamicum,fungi, algae, or ciliates on stress tolerance can be assessed by growingthe modified microorganism or plant under less than suitable conditionsand then analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

[0119] For example, yeast expression vectors comprising the nucleicacids disclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived there from can then be assayedfor fail or alteration of their tolerance to drought, salt, andtemperature stress.

[0120] The engineering of one or more CCSRP genes of the invention mayalso result in CCSRPs having altered activities which indirectly impactthe stress response and/or stress tolerance of algae, plants, ciliatesor fungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more CCSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

[0121] Additionally, the sequences disclosed herein, or fragmentsthereof, can be used to generate knockout mutations in the genomes ofvarious organisms, such as bacteria, mammalian cells, yeast cells, andplant cells (Girke, T., 1998 The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation see U.S. Pat. No.6004804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

[0122] The aforementioned mutagenesis strategies for CCSRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated CCSRP nucleic acidand protein molecules such that the stress tolerance is improved.

[0123] The present invention also provides antibodies that specificallybind to a CCSRP, or a portion thereof, as encoded by a nucleic aciddescribed herein. Antibodies can be made by many well-known methods(See, e.g. Harlow and Lane, “Antibodies; A Laboratory Manual” ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly,purified antigen can be injected into an animal in an amount and inintervals sufficient to elicit an immune response. Antibodies can eitherbe purified directly, or spleen cells can be obtained from the animal.The cells can then fused with an immortal cell line and screened forantibody secretion. The antibodies can be used to screen nucleic acidclone libraries for cells secreting the antigen. Those positive clonescan then be sequenced. (See, for example, Kelly et al., 1992Bio/Technology 10:163-167; Bebbington et al., 1992 Bio/Technology10:169-175).

[0124] The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding of an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies that selectivelybind with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies selectivelyimmunoreactive with a protein. See Harlow and Lane “Antibodies, ALaboratory Manual” Cold Spring Harbor Publications, New York, (1988),for a description of immunoassay formats and conditions that could beused to determine selective binding.

[0125] In some instances, it is desirable to prepare monoclonalantibodies from various hosts. A description of techniques for preparingsuch monoclonal antibodies may be found in Stites et al., editors,“Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos,Calif., Fourth Edition) and references cited therein, and in Harlow andLane (“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York, 1988).

[0126] Throughout this application, various publications are referenced.The disclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

[0127] It should also be understood that the foregoing relates topreferred embodiments of the present invention and that numerous changesmay be made therein without departing from the scope of the invention.The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example 1 Growth of Physcomitrella patens Cultures

[0128] For this study, plants of the species Physcomitrella patens(Hedw.) B. S. G. from the collection of the genetic studies section ofthe University of Hamburg were used. They originate from the strain16/14 collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

[0129] Culturing was carried out in a climatic chamber at an airtemperature of 25° C. and light intensity of 55 micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube) and a light/dark change of16/8 hours. The moss was either modified in liquid culture using Knopmedium according to Reski and Abel (1985, Planta 165:354-358) orcultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke,England). The protonemas used for RNA and DNA isolation were cultured inaerated liquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2 Total DNA Isolation from Plants

[0130] The details for the isolation of total DNA relate to the workingup of one gram fresh weight of plant material. The materials usedinclude the following buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

[0131] The plant material was triturated under liquid nitrogen in amortar to give a fine powder and transferred to 2 ml Eppendorf vessels.The frozen plant material was then covered with a layer of 1 ml ofdecomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosinebuffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K solution,10 mg/ml) and incubated at 60° C. for one hour with continuous shaking.The homogenate obtained was distributed into two Eppendorf vessels (2ml) and extracted twice by shaking with the same volume ofchloroform/isoamyl alcohol (24:1). For phase separation, centrifugationwas carried out at 8000×g and room temperature for 15 minutes in eachcase. The DNA was then precipitated at −70° C. for 30 minutes usingice-cold isopropanol. The precipitated DNA was sedimented at 4° C. and10,000 g for 30 minutes and resuspended in 180 μl of TE buffer (Sambrooket al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).For further purification, the DNA was treated with NaCl (1.2 M finalconcentration) and precipitated again at −70° C. for 30 minutes usingtwice the volume of absolute ethanol. After a washing step with 70%ethanol, the DNA was dried and subsequently taken up in 50 μl ofH₂O+RNAse (50 mg/ml final concentration). The DNA was dissolvedovernight at 4° C. and the RNAse digestion was subsequently carried outat 37° C. for 1 hour. Storage of the DNA took place at 4° C.

Example 3 Isolation of Total RNA and Poly-(A)+ RNA and cDNA LibraryConstruction from Physcomitrella patens

[0132] For the investigation of transcripts, both total RNA andpoly-(A)⁺ RNA were isolated. The total RNA was obtained from wild-type 9day old protonemata following the GTC-method (Reski et al. 1994, Mol.Gen. Genet., 244:352-359). The Poly(A)+ RNA was isolated using DynaBeadsR (Dynal, Oslo, Norway) following the instructions of themanufacturers protocol. After determination of the concentration of theRNA or of the poly(A)+ RNA, the RNA was precipitated by addition of{fraction (1/10)} volumes of 3 M sodium acetate pH 4.6 and 2 volumes ofethanol and stored at −70° C.

[0133] For cDNA library construction, first strand synthesis wasachieved using Murine Leukemia Virus reverse transcriptase (Roche,Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis byincubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour). The reaction wasstopped by incubation at 65° C. (10 minutes) and subsequentlytransferred to ice. Double stranded DNA molecules were blunted byT4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotideswere removed by phenol/chloroform extraction and Sephadex G50 spincolumns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated tothe cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) andphosphorylated by incubation with polynucleotide kinase (Roche, 37° C.,30 minutes). This mixture was subjected to separation on a low meltingagarose gel. DNA molecules larger than 300 base pairs were eluted fromthe gel, phenol extracted, concentrated on Elutip-D-columns (Schleicherand Schuell, Dassel, Germany) and were ligated to vector arms and packedinto lambda ZAPII phages or lambda ZAP-Express phages using the GigapackGold Kit (Stratagene, Amsterdam, Netherlands) using material andfollowing the instructions of the manufacturer.

Example 4 Sequencing and Function Annotation of Physcomitrella patensESTs

[0134] cDNA libraries as described in Example 3 were used for DNAsequencing according to standard methods, and in particular, by thechain termination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used: 5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO: 105′-CTAAAGGGAACAAAAGCTG-3′ SEQ ID NO: 11 5′-TGTAAAACGACGGCCAGT-3′ SEQ IDNO: 12

[0135] Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpg.de. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; Pearson W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P. (1997) 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTALW: Multiplesequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994) CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680; TMAP: Transmembrane region prediction from multiplyaligned sequences. Persson, B. and Argos, P. (1994) Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C. Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M.,Smith J. E. (1992) ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function.Biotechniques 13, 919-921; BLIMPS: Similarity searches against adatabase of ungapped blocks. J. C. Wallace and Henikoff S., (1992);PATMAT: A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5 Identification of Physcomitrella patens ORFs Corresponding toCC-1, CC-2 and CC-3

[0136] The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1below were identified in the Physcomitrella patens EST sequencingprogram using the program EST-MAX through BLAST analysis. The SequenceIdentification Numbers corresponding to these ESTs are as follows: CC-1(SEQ ID NO: 1), CC-2 (SEQ ID NO:2) and CC-3 (SEQ ID NO:3). TABLE 1Functional ORF Name categories Function Sequence code position PpCC-1Cell Cycle suppressor protein c_pp004062017r 116-886 PpCC-2 Cell Cyclecell division control c_pp004064149r  1-590 protein PpCC-3 Cell Cyclecell division s_pp002007053r 106-492 protein

[0137] TABLE 2 Degree of Amino Acid Identity and Similarity of PpCC-1and Other Homologous Proteins GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9ZNT0P46467 P52917 Q9UI03 Q09803 Protein name PUTATIVE SKD1 SKD1 PROTEINVACUOLAR SKD1 PROTEIN SUPPRESSOR PROTEIN PROTEIN PROTEIN OF SORTING-BEM1/BED5 ASSOCIATED DOUBLE PROTEIN VPS4 MUTANTS Species Arabidopsis Musmusculus Saccharomyces Homo sapiens Schizosaccharo- thaliana (Mouse)cerevisiae (Human) myces pombe (Mouse-ear cress) (Baker's yeast)(Fission yeast) Identity % 82% 54% 55% 53% 52% Similarity % 90% 66% 67%65% 65%

[0138] TABLE 3 Degree of Amino Acid Identity and Similarity of PpCC-2and Other Homologous Proteins GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9SIV8P29458 P30665 P49717 P33991 Protein name PUTATIVE CDC21 PROTEIN CELLDIVISION DNA REPLICATION DNA REPLICATION CDC21 CONTROL LICENSING FACTORLICENSING FACTOR PROTEIN PROTEIN 54 MCM4 MCM4 Species ArabidopsisSchizo- Saccharomyces Mus musculus Homo sapiens thaliana saccharomycescerevisiae (Mouse) (Human) (Mouse-ear pombe (Baker's yeast) cress)(Fission yeast) Identity % 49% 41% 40% 43% 43% Similarity % 61% 55% 54%56% 57%

[0139] TABLE 4 Degree of Amino Acid Identity and Similarity of PpCC-3and Other Homologous Proteins GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # O80860Q9LXF7 P51327 O78516 Q55700 Protein name PUTATIVE CELL FTSH-LTKE CELLDIVISION CELL DIVISION CELL DIVISION DIVISION PROTEIN PROTEIN PFTFPROTEIN FTSH PROTEIN FTSH PROTEIN FTSH (ZINC DEPENDENT HOMOLOG HOMOLOGHOMOLOG 1 PROTEASE) Species Arabidopsis Arabidopsis Porphyra Guillardiatheta Synechocyst is sp. thaliana thaliana purpurea (Cryptomonas phi)(strain PCC 6803) (Mouse-ear (Mouse-ear cress) cress) Identity % 77% 64%60% 59% 56% Similarity % 84% 75% 69% 69% 67%

Example 6 Cloning of the Full-Length Physcomitrella patens cDNA Encodingfor CC-1, CC-2 and CC-3

[0140] To isolate the clones encoding Pp CC-1 (SEQ ID NO:4), PpCC-2 (SEQID NO:5) and PpCC-3 (SEQ ID NO:6) from Physcomitrella patens, cDNAlibraries were created with SMART RACE cDNA Amplification kit (ClontechLaboratories) following manufacturer's instructions. Total RNA isolatedas described in Example 3 was used as the template. The cultures weretreated prior to RNA isolation as follows: Salt Stress: 2, 6, 12, 24, 48hours with 1-M NaC1-supplemented medium; Cold Stress: 4° C. for the sametime points as for salt; Drought Stress: cultures were incubated on dryfilter paper for the same time points as for salt.

[0141] 5′ RACE Protocol

[0142] The EST sequences CC-2 (SEQ ID NO:2) and CC-3 (SEQ ID NO:3)identified from the database search as described in Example 4 were usedto design oligos for RACE (see Table 5). The extended sequences forthese genes were obtained by performing Rapid Amplification of cDNA Endspolymerase chain reaction (RACE PCR) using the Advantage 2 PCR kit(Clontech Laboratories) and the SMART RACE cDNA amplification kit(Clontech Laboratories) using a Biometra T3 Thermocycler following themanufacturer's instructions. The sequences obtained from the RACEreactions corresponded to full-length coding regions of CC-2 and CC-3and were used to design oligos for full-length cloning of the respectivegenes (see below full-length amplification).

[0143] Full-Length Amplification

[0144] Full-length clones corresponding CC-1 (SEQ ID NO:4), CC-2 (SEQ IDNO:5), and CC-3 (SEQ ID NO:6) were obtained by performing polymerasechain reaction (PCR) with gene-specific primers (see Table 5) and theoriginal EST as the template. The conditions for the reaction werestandard conditions with PWO DNA polymerase (Roche). PCR was performedaccording to standard conditions and to manufacturer's protocols(Sambrook et al., 1989 Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,Biometra T3 Thermocycler). The parameters for the reaction were: fiveminutes at 94° C. followed by five cycles of one minute at 94° C., oneminute at 50° C. and 1.5 minutes at 72° C. This was followed by twentyfive cycles of one minute at 94° C., one minute at 65° C. and 1.5minutes at 72° C.

[0145] The amplified fragments were extracted from agarose gel with aQIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1vector (Invitrogen) following manufacturer's instructions. Recombinantvectors were transformed into Top10 cells (Invitrogen) using standardconditions (Sambrook et al. 1989. Molecular Cloning, A LaboratoryManual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold SpringHarbor, N.Y.). Transformed cells were selected for on LB agar containing100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonieswere selected and used to inoculate 3 ml of liquid LB containing 100μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extractedusing the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. Analyses of subsequent clones and restriction mapping wasperformed according to standard molecular biology techniques (Sambrooket al., 1989 Molecular Cloning, A Laboratory Manual. 2nd Edition. ColdSpring Harbor Laboratory Press. Cold Spring Harbor, N.Y.). TABLE 5Scheme and primers used for cloning of full-length clones Sites in Genefinal product Isolation Method Primers Race Primer RT-PCR PpCC-1EcoRV/SacI 5′ RACE and N/A RC409 RT-PCR for (SEQ ID NO: 13) Full-lengthGCGATATCGACC clone CAAGGTGTGTAG AGAAGGGGAT RC410 (SEQ ID NO: 14)GCGAGCTCGCGA CTGATGCAACTCA CCCATGAA PpCC-2 AscI/SacI 5′ RACE and RC793:RC990: RT-PCR for (SEQ ID NO: 15) (SEQ ID NO: 18) Full-lengthCCCAAATGCTT ATGGCGCGCCGC clone GGAGCCAGCGA ACTCACGTGAGG CCT AATTGCACCTCCRC794: RC991: (SEQ ID NO: 16) (SEQ ID NO: 19) GACGTGCACGC GCGAGCTCTGGGAATCGATGTAG AATTGCGCATGG GTC GAAACTGG NVT: (SEQ ID NO: 17) AGTCGACCCCTGTCAGACTTCTT GA PpCC-3 XmaI/SacI 5′ RACE and NVT: RC695: RT-PCR for (SEQID NO: 20) (SEQ ID NO: 21) Full-length CTGGCTACCCA ATCCCGGGTGAT cloneAACCGCGTCGG ACGTTGTGCATAT AGA TTGGTGTTGC RC696: (SEQ ID NO: 22)GCGAGCTCTGAT ACAAGAGCGTCG ATGCTCCA

Example 7 Engineering Stress-Tolerant Arabidopsis Plants byOver-Expressing the Genes CC-1, CC-2 and CC-3

[0146] Binary Vector Construction: Kanamycin

[0147] The plasmid construct pACGH101 was digested with PstI (Roche) andFseI (NEB) according to manufacturers' instructions. The fragment waspurified by agarose gel and extracted via the Qiaex II DNA Extractionkit (Qiagen). This resulted in a vector fragment with the ArabidopsisActin2 promoter with internal intron and the OCS3 terminator. Primersfor PCR amplification of the NPTII gene were designed as follows:5′NPT-Pst: GCG-CTG-CAG-ATT-TCA-TTT-GGA-GAG- (SEQ ID NO: 23) GAC-ACG3′NPT-Fse: CGC-GGC-CGG-CCT-CAG-AAG-AAC-TCG- (SEQ ID NO: 24)TCA-AGA-AGG-CG

[0148] The 0.9 kilobase NPTII gene was amplified via PCR from pCambia2301 plasmid DNA [94° C. 60 sec, {94° C. 60 sec, 61° C. (−0.1° C. percycle) 60 sec, 72° C. 2 min}×25 cycles, 72° C. 10 min on BiometraT-Gradient machine], and purified via the Qiaquick PCR Extraction kit(Qiagen) as per manufacturer's instructions. The PCR DNA was thensubcloned into the pCR-BluntII TOPO vector (Invitrogen) pursuant to themanufacturer's instructions (NPT-Topo construct). These ligations weretransformed into Top10 cells (Invitrogen) and grown on LB plates with 50ug/ml kanamycin sulfate overnight at 37° C. Colonies were then used toinoculate 2 ml LB media with 50 ug/ml kanamycin sulfate and grownovernight at 37° C. Plasmid DNA was recovered using the Qiaprep SpinMiniprep kit (Qiagen) and sequenced in both the 5′ and 3′ directionsusing standard conditions. Subsequent analysis of the sequence datausing VectorNTI software revealed no PCR errors present in the NPTIIgene sequence.

[0149] The NPT-Topo construct was then digested with PstI (Roche) andFseI (NEB) according to manufacturers' instructions. The 0.9 kilobasefragment was purified on agarose gel and extracted by Qiaex II DNAExtraction kit (Qiagen). The Pst/Fse insert fragment from NPT-Topo andthe Pst/Fse vector fragment from pACGH101 were then ligated togetherusing T4 DNA Ligase (Roche) following manufacturer's instructions. Theligation was then transformed into Top10 cells (Invitrogen) understandard conditions, creating pBPSsc019 construct. Colonies wereselected on LB plates with 50 μg/ml kanamycin sulfate and grownovernight at 37° C. These colonies were then used to inoculate 2ml LBmedia with 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)following the manufacturer's instructions.

[0150] The pBPSSC019 construct was digested with KpnI and BsaI (Roche)according to manufacturer's instructions. The fragment was purified viaagarose gel and then extracted via the Qiaex II DNA Extraction kit(Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPTcassette, which included the Arabidopsis Actin2 promoter with internalintron, the NPTII gene and the OCS3 terminator.

[0151] The pBPSJH001 vector was digested with SpeI and ApaI (Roche) andblunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) accordingto manufacture's instructions. This produced a 10.1 kilobase vectorfragment minus the Gentamycin cassette, which was recircularized byself-ligating with T4 DNA Ligase (Roche), and transformed into Top10cells (Invitrogen) via standard conditions. Transformed cells wereselected for on LB agar containing 50 μg/ml kanamycin sulfate and grownovernight at 37° C. Colonies were then used to inoculate 2 ml of liquidLB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen)following manufacture's instructions. The recircularized plasmid wasthen digested with KpnI (Roche) and extracted from agarose gel via theQiaex II DNA Extraction kit (Qiagen) as per manufacturers' instructions.

[0152] The Act-NPT Kpn-cut insert and the Kpn-cut pBPSJH001recircularized vector were then ligated together using T4 DNA Ligase(Roche) and transformed into Top10 cells (Invitrogen) as permanufacturers' instructions. The resulting construct, pBPSsc022, nowcontained the Super Promoter, the GUS gene, the NOS terminator, and theAct-NPT cassette. Transformed cells were selected for on LB agarcontaining 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Colonies were then used to inoculate 2 ml of liquid LB containing 50μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA wasextracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. After confirmation of ligation success viarestriction digests, pBPSsc022 plasmid DNA was further propagated andrecovered using the Plasmid Midiprep Kit (Qiagen) following themanufacturer's instructions.

[0153] Subcloning of CC-1, CC-2 and CC-3 into the Binary Vector

[0154] The fragments containing the different Physcomitrella patenstranscription factors were subcloned from the recombinant PCR2.1 TOPOvectors by double digestion with restriction enzymes (see Table 6)according to manufacturer's instructions. The subsequence fragment wasexcised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen)according to manufacture's instructions and ligated into the binaryvectors pGMSG, cleaved with XmaI and Ec1136II and dephosphorylated priorto ligation. The resulting recombinant pGMSG contained the correspondingtranscription factor in the sense orientation under the constitutivesuper promoter. TABLE 6 Listed are the names of the various constructsof the Physcomitrella patens transcription factors used for planttransformation Enzymes used to generate gene Enzymes used to BinaryVector Gene fragment restrict pBPSJH001 Construct PpCC-1 EcoRV/SacISmaI/SacI pBPSSY032 PpCC-2 AscI/SacI XmaI/SacI pBPSsc335 PpCC-3XmaI/SacI XmaI/SacI pBPSLVM186

[0155] Agrobacterium Transformation

[0156] The recombinant vectors were transformed into Agrobacteriumtumefaciens C58C1 and PMP90 according to standard conditions (Hoefgenand Willmitzer, 1990).

[0157] Plant Transformation

[0158]Arabidopsis thaliana ecotype C24 were grown and transformedaccording to standard conditions (Bechtold 1993, Acad. Sci. Paris.316:1194-1199; Bent et al. 1994, Science 265:1856-1860).

[0159] Screening of Transformed Plants

[0160] T1 seeds were sterilized according to standard protocols (Xionget al. 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds wereplated on ½ MS 0.6% agar supplemented with 1% sucrose, 50 μg/mlkanamycin (Sigma-Aldrich) and 2 μg/ml benomyl (Sigma-Aldrich). Seeds onplates were vernalized for four days at 4° C. The seeds were germinatedin a climatic chamber at an air temperature of 22° C. and lightintensity of 40 micromols^(−1m2) (white light; Philips TL 65W/25fluorescent tube) and 16 hours light and 8 hours dark day length cycle.Transformed seedlings were selected after 14 days and transferred to ½MS 0.6% agar plates supplemented with 1% sucrose and allowed to recoverfor five-seven days.

[0161] Drought Tolerance Screening

[0162] T1 seedlings were transferred to dry, sterile filter paper in apetri dish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Sanyo Growth Cabinet MLR-350H, micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube). The RH was then decreased to60% and the seedlings were desiccated further for eight hours. Seedlingswere then removed and placed on ½ MS 0.6% agar plates supplemented with2 μg/ml benomyl and scored after five days.

[0163] Under drought stress conditions, PpCC-1 over-expressingArabidopsis thaliana plants showed a 67% (6 survivors from 9 stressedplants) survival rate to the stress screening; PpCC-2, 75% (6 survivorsfrom 8 stressed plants) and PpCC-3, 92% (11 survivors from 12 stressedplants), whereas the control only showed a 11% survival rate (1 survivorfrom 9 stressed plants) (see Table 7). It is noteworthy that theanalyses of these transgenic lines were performed with T1 plants, andtherefore, the results will be better when a homozygous, strongexpresser is found. TABLE 7 Summary of the drought stress tests DroughtStress Test Gene Number of Total number of Percentage of Name survivorsplants survivors PpCC-1 6 9 67% PpCC-2 6 8 75% PpCC-3 11 12 92%

[0164] Freezing Tolerance Screening

[0165] Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing −1° C. hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days.

[0166] Under freezing stress conditions, PpCC-1 over-expressingArabidopsis thaliana plants showed a 89% (8 survivors from 9 stressedplants) survival rate to the stress screening, whereas the untransformedcontrol only showed a 2% survival rate (1 survivor from 48 stressedplants) (see Table 8). It is noteworthy that the analyses of thesetransgenic lines were performed with T1 plants, and therefore, theresults will be better when a homozygous, strong expresser is found.TABLE 8 Summary of the freezing stress tests Freezing Stress Test GeneTotal number of Percentage of Name Number of survivors plants survivorsPpCC-1 8  9 89% Control 1 48  2%

[0167] Salt Tolerance Screening

[0168] Seedlings were transferred to filter paper soaked in ½ MS andplaced on ½ MS 0.6% agar supplemented with 2 μg/ml benomyl the nightbefore the salt tolerance screening. For the salt tolerance screening,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 50 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked with 200 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 600 mM NaCl, in a petri dish. After 10 hours,the seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2 μg/ml benomyl. The seedlings were scored after 5days.

[0169] The transgenic plants are screened for their improved salttolerance demonstrating that transgene expression confers salttolerance.

Example 8 Detection of the CC-1, CC-2 and CC-3 Transgenes in theTransgenic Arabidopsis Lines

[0170] One leaf from a wild type and a transgenic Arabidopsis plant washomogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB) buffer(2% CTAB, 1.4 M NaCl, 8 mM EDTA and 20 mM Tris pH 8.0) and 1 μlβ-mercaptoethanol. The samples were incubated at 60-65° C. for 30minutes and 250 μl of Chloroform was then added to each sample. Thesamples were vortexed for 3 minutes and centrifuged for 5 minutes at18,000×g. The supernatant was taken from each sample and 150 μlisopropanol was added. The samples were incubated at room temperaturefor 15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pelletwas washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4 μl ofabove suspension was used in a 20 μl PCR reaction using Taq DNApolymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions. The primers used in the reactions were:PpCC-1 GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO: 25)GCGAGCTCGCGACTGATGCAACTCACCCATGAA (SEQ ID NO: 26)

[0171] The PCR program was as following: 30 cycles of 1 minute at 94°C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutesat 72° C. A 1.6 kb fragment was produced from the positive control andthe transgenic plants. PpCC-2 GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO: 25)GCGAGCTCTGGGAATTGCGCATGGGAAACTGG (SEQ ID NO: 27)

[0172] The primers were used in the first round of reactions with thefollowing program: 30 cycles of 1 minute at 94° C., 1 minute at 62° C.and 4 minutes at 72° C., 10 minutes at 72° C. A 3 kb fragment wasgenerated from the positive control and the T1 transgenic plants. PpCC-3GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO: 25)GCGAGCTCTGATACAAGAGCGTCGATGCTCCA (SEQ ID NO: 28)

[0173] The PCR program was as following: 30 cycles of 1 minute at 94°C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutesat 72° C. A 2.3 kb fragment was produced from the positive control andthe transgenic plants.

[0174] The transgenes were successfully amplified from the T1 transgeniclines, but not from the wild type C24. This result indicates that the T1transgenic plants contain at least one copy of the transgenes. There wasno indication of existence of either identical or very similar genes inthe untransformed Arabidopsis thaliana control that could be amplifiedby this method.

Example 9 Detection of the CC-1, CC-2 and CC-3 Transgene mRNA inTransgenic Arabidopsis Lines

[0175] Transgene expression was detected using RT-PCR. Total RNA wasisolated from stress-treated plants using a procedure adapted from(Verwoerd et al., 1989 NAR 17:2362). Leaf samples (50-100 mg) werecollected and ground to a fine powder in liquid nitrogen. Ground tissuewas resuspended in 500 μl of a 80° C., 1:1 mixture, of phenol toextraction buffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS),followed by brief vortexing to mix. After the addition of 250 μl ofchloroform, each sample was vortexed briefly. Samples were thencentrifuged for 5 minutes at 12,000×g. The upper aqueous phase wasremoved to a fresh eppendorf tube. RNA was precipitated by adding{fraction (1/10)}^(th) volume 3M sodium acetate and 2 volumes 95%ethanol. Samples were mixed by inversion and placed on ice for 30minutes. RNA was pelleted by centrifugation at 12,000×g for 10 minutes.The supernatant was removed and pellets briefly air-dried. RNA samplepellets were resuspended in 10 μl DEPC treated water. To removecontaminating DNA from the samples, each was treated with RNase-freeDNase (Roche) according to the manufacturer's recommendations. cDNA wassynthesized from total RNA using the 1^(st) Strand cDNA synthesis kit(Boehringer Mannheim) following manufacturer's recommendations.

[0176] PCR amplification of a gene-specific fragment from thesynthesized cDNA was performed using Taq DNA polymerase (Roche) andgene-specific primers (see below for primers) in the following reaction:1×PCR buffer, 1.5 mM MgCl₂, 0.2 μM each primer, 0.2 μM dNTPs, 1 unitpolymerase, 5 μl cDNA from synthesis reaction. Amplification wasperformed under the following conditions: Denaturation, 95° C., 1minute; annealing, 62° C., 30 seconds; extension, 72° C., 1 minute, 35cycles; extension, 72° C., 5 minutes; hold, 4° C., forever. PCR productswere run on a 1% agarose gel, stained with ethidium bromide, andvisualized under UV light using the Quantity-One gel documentationsystem (Bio-Rad).

[0177] Expression of the transgenes was detected in the T1 transgenicline. This result indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic line. On the other hand, noexpression of identical or very similar endogenous genes could bedetected by this method. These results are in agreement with the datafrom Example 7. This greatly supports our statement that the observedstress tolerance is due to the introduced transgene.

[0178] The following primer combination was used in the above-describedreactions. PpCC-1: GATGGTGATGGTGACAGCGAGGATC (SEQ ID NO: 29)CGACGTGAAGCCTCGCTCTCATTTCC (SEQ ID NO: 30) PpCC-2:CGATCTCCTTCAAGGGAACCTAGTGCTG (SEQ ID NO: 31) GAGTTCACGCATGTGCACCGATGCT(SEQ ID NO: 32) PpCC-3: CGTAGCGTCTTCCAGGATGTCCTAC (SEQ ID NO: 33)GCTTGGCCTCGTCTACTCCCGCAAC (SEQ ID NO: 34)

[0179] Amplification was performed under the following conditions:Denaturation, 95° C., 1 minute; annealing, 62° C., 30 seconds;extension, 72° C., I minute, 35 cycles; extension, 72° C., 5 minutes;hold, 4° C., forever. PCR products were run on a 1% agarose gel, stainedwith ethidium bromide, and visualized under UV light using theQuantity-One gel documentation system (Bio-Rad).

[0180] Expression of the transgenes was detected in the T1 transgenicline. These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7.

Example 10 Engineering Stress-Tolerant Soybean Plants by Over-Expressingthe CC-1, CC-2 and CC-3 Gene

[0181] The constructs pBPSSY032, pBPSsc335 and pBPSLVM186 were used totransform soybean as described below.

[0182] Seeds of soybean were surface sterilized with 70% ethanol for 4minutes at room temperature with continuous shaking, followed by 20%(v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds were rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats were peeled off, andcotyledons are detached from the embryo axis. The embryo axis wasexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes were collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

[0183]Agrobacterium tumefaciens culture was prepared from a singlecolony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/lstreptomycin, 50 mg/l kanamycin) followed by growth of the single colonyin liquid LB medium to an optical density at 600 nm of 0.8. Then, thebacteria culture was pelleted at 7000 rpm for 7 minutes at roomtemperature, and resuspended in MS (Murashige and Skoog, 1962) mediumsupplemented with 100 μM acetosyringone. Bacteria cultures wereincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately15% moisture content were imbibed for 2 hours at room temperature withthe pre-induced Agrobacterium suspension culture. The embryos areremoved from the imbibition culture and were transferred to Petri dishescontaining solid MS medium supplemented with 2% sucrose and incubatedfor 2 days, in the dark at room temperature. Alternatively, the embryoswere placed on top of moistened (liquid MS medium) sterile filter paperin a Petri dish and incubated under the same conditions described above.After this period, the embryos were transferred to either solid orliquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/Lcefotaxime to kill the agrobacteria. The liquid medium was used tomoisten the sterile filter paper. The embryos were incubated during 4weeks at 25° C., under 150 μmol m⁻²sec⁻¹ and 12 hours photoperiod. Oncethe seedlings produced roots, they were transferred to sterile metromixsoil. The medium of the in vitro plants was washed off beforetransferring the plants to soil. The plants were kept under a plasticcover for 1 week to favor the acclimatization process. Then the plantswere transferred to a growth room where they were incubated at 25° C.,under 150 μmol m⁻²sec⁻¹ light intensity and 12 hours photoperiod forabout 80 days.

[0184] The transgenic plants were then screened for their improveddrought, salt and/or cold tolerance according to the screening methoddescribed in Example 7 demonstrating that transgene expression confersstress tolerance.

Example 11 Engineering Stress-Tolerant Rapeseed/Canola Plants byOver-Expressing the CC-1, CC-2 and CC-3 Genes

[0185] The constructs pBPSSY032, pBPSsc335 and pBPSLVM186 were used totransform rapseed/canola as described below.

[0186] The method of plant transformation described herein is alsoapplicable to Brassica and other crops. Seeds of canola are surfacesterilized with 70% ethanol for 4 minutes at room temperature withcontinuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05%(v/v) Tween for 20 minutes, at room temperature with continuous shaking.Then, the seeds are rinsed 4 times with distilled water and placed onmoistened sterile filter paper in a Petri dish at room temperature for18 hours. Then the seed coats are removed and the seeds are air driedovernight in a half-open sterile Petri dish. During this period, theseeds lose approx. 85% of its water content. The seeds are then storedat room temperature in a sealed Petri dish until further use. DNAconstructs and embryo imbibition are as described in Example 10. Samplesof the primary transgenic plants (T0) are analyzed by PCR to confirm thepresence of T-DNA. These results are confirmed by Southern hybridizationin which DNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

[0187] The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7demonstrating that transgene expression confers drought tolerance.

Example 12 Engineering Stress-Tolerant Corn Plants by Over-Expressingthe CC-1, CC-2 and CC-3 Genes

[0188] The constructs pBPSSY032, pBPSsc335 and pBPSLVM186 were used totransform corn as described below.

[0189] Transformation of maize (Zea Mays L.) is performed with themethod described by Ishida et al. 1996. Nature Biotch 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt and/or cold tolerance according to the screeningmethod described in Example 7 demonstrating that transgene expressionconfers stress tolerance.

Example 13 Engineering Stress-Tolerant Wheat Plants by Over-Expressingthe CC-1, CC-2 and CC-3 Genes

[0190] The constructs pBPSSY032, pBPSsc335 and pBPSLVM186 were used totransform wheat as described below.

[0191] Transformation of wheat is performed with the method described byIshida et al. 1996 Nature Biotch. 14745-50. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors, and transgenic plants are recovered through organogenesis. Thisprocedure provides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 14 Identification of Homologous and Heterologous Genes

[0192] Gene sequences can be used to identify homologous or heterologousgenes from cDNA or genomic libraries. Homologous genes (e. g.full-length cDNA clones) can be isolated via nucleic acid hybridizationusing for example cDNA libraries. Depending on the abundance of the geneof interest, 100,000 up to 1,000,000 recombinant bacteriophages areplated and transferred to nylon membranes. After denaturation withalkali, DNA is immobilized on the membrane by e. g. UV cross linking.Hybridization is carried out at high stringency conditions. In aqueoussolution hybridization and washing is performed at an ionic strength of1 M NaCl and a temperature of 68° C. Hybridization probes are generatedby e. g. radioactive (32p) nick transcription labeling (High Prime,Roche, Mannheim, Germany). Signals are detected by autoradiography.

[0193] Partially homologous or heterologous genes that are related butnot identical can be identified in a manner analogous to theabove-described procedure using low stringency hybridization and washingconditions. For aqueous hybridization, the ionic strength is normallykept at 1 M NaCl while the temperature is progressively lowered from 68to 42° C.

[0194] Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

[0195] Oligonucleotide hybridization solution:

[0196] 6×SSC

[0197] 0.01 M sodium phosphate

[0198] 1 mM EDTA (pH 8)

[0199] 0.5% SDS

[0200] 100 μg/ml denatured salmon sperm DNA

[0201] 0.1% nonfat dried milk

[0202] During hybridization, temperature is lowered stepwise to 5-10° C.below the estimated oligonucleotide Tm or down to room temperaturefollowed by washing steps and autoradiography. Washing is performed withlow stringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al. (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.

Example 15 Identification of Homologous Genes by Screening ExpressionLibraries with Antibodies

[0203] c-DNA clones can be used to produce recombinant protein forexample in E. coli (e. g. Qiagen QIAexpress pQE system). Recombinantproteins are then normally affinity purified via Ni—NTA affinitychromatography (Qiagen). Recombinant proteins are then used to producespecific antibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni—NTA columnsaturated with the recombinant antigen as described by Gu et al., 1994BioTechniques 17:257-262. The antibody can than be used to screenexpression cDNA libraries to identify homologous or heterologous genesvia an immunological screening (Sambrook, J. et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 16 In Vivo Mutagenesis

[0204] In vivo mutagenesis of microorganisms can be performed by passageof plasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M. (1994)Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 17 In Vitro Analysis of the Function of Physcomitrella Genes inTransgenic Organisms

[0205] The determination of activities and kinetic parameters of enzymesis well established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh, (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.VCH: Weinheim, p. 352-363.

[0206] The activity of proteins which bind to DNA can be measured byseveral well-established methods, such as DNA band-shift assays (alsocalled gel retardation assays). The effect of such proteins on theexpression of other molecules can be measured using reporter gene assays(such as that described in Kolmar, H. et al. (1995) EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems arewell known and established for applications in both pro- and eukaryoticcells, using enzymes such as β-galactosidase, green fluorescent protein,and several others.

[0207] The determination of activity of membrane-transport proteins canbe performed according to techniques such as those described in Gennis,R.B. Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg (1989).

Example 18 Purification of the Desired Product from TransformedOrganisms

[0208] Recovery of the desired product from plant material (i.e.,Physcomitrella patents or Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, canbe harvested from the culture by low-speed centrifugation, the cells canbe lysed by standard techniques, such as mechanical force orsonification. Organs of plants can be separated mechanically from othertissue or organs. Following homogenization cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and the supernatefraction is retained for further purification.

[0209] The supernatant fraction from either purification method issubjected to chromatography with a suitable resin, in which the desiredmolecule is either retained on a chromatography resin while many of theimpurities in the sample are not, or where the impurities are retainedby the resin while the sample is not. Such chromatography steps may berepeated as necessary, using the same or different chromatographyresins. One skilled in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

[0210] There is a wide array of purification methods known to the artand the preceding method of purification is not meant to be limiting.Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986). Additionally, the identity and purity of the isolatedcompounds may be assessed by techniques standard in the art. Theseinclude high-performance liquid chromatography (HPLC), spectroscopicmethods, staining methods, thin layer chromatography, NIRS, enzymaticassay, or microbiologically. Such analysis methods are reviewed in:Patek et al., 1994 Appl. Environ. Microbiol. 60:133-140; Malakhova etal., 1996 Biotekhnologiya 11:27-32; and Schmidt et al., 1998 BioprocessEngineer. 19:67-70. Ulmann's Encyclopedia of Industrial Chemistry,(1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p.559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

APPENDIX

[0211] Nucleotide sequence of the partial CC-1 from Physcomitrellapatens (SEQ ID NO:1)CGGGAGTTGGTGATCTTCAGCATGCTCGTAGTTGACAAGGGAGGTAGGATTGTTTGACCCAAGGTGTGTANAGAAGGGGATAGCCATGTACAGCAACTTCAAAGAGCAGGCCATAGAATATTGTGCGTCAAGCCGTAGCGGAAGACAACGCAGGGAACTATGCCAAAGCGTTTCCGCTGTACATGAACGCGCTTGAGTACTTCAAGACGCATCTAAAGTATGAGAAGAATCCCAAAATCAAGGAGGCCATCACTCAGAAGTTCACGGAGTATTTGAGGAGGGCGGAGGAGATTCGAGCCGTTTTGGACGATGGCCCCACTGGACCCTCTGCAAATGGAGACGCGGCAGTTCAAGCTAAACCGAAGTCGAAATCAGGGAAGAAGGATGGTGGCGGGGGTGATGGTGATGGTGACAGCGAGGATCCCGACCAGCAGAAGCTGAGATCAGGGCTGAACTCGGCAATCATACGGGAAAAGCCAAATGTTCGGTGGGCTGATGTTGCTGGACTTGAAAGTGCCAAGCAGGCGTTGCAGGAGGCAGTGATCTTGCCCGTGAAGTTTCCCCAATTTTTCACAGGGAAGCGAAGAACATGGCGAGCATTTTTGTGGTATGGGCCCCCCGGGACTGGAAAATCGTATCTTGCAAAAGCTGTTGCTACGGAAGCTGATTCTACATTCTTTAGTATTTCCTCTTCAGACTTGGTGTCAAAGTGGATGGGAGAGAGTGAGAAGCTTGTTGCAAATCTGTTTCAAATGGCCCGTGAAGCTGCTCCATCCATCATCTTCATAGACGAGATTGATTCTTTATGCGGTACTCGAGGTGAAGGAAATGAGAGCGAGGCTTCACGTCGTATCAAGACTGAGTTGCTAGTTCAAATG CAGGGTGTC

[0212] Nucleotide sequence of the partial CC-2 from Physcomitrellapatens (SEQ ID NO:2)CGGCACCAGGGAGATCGTATTGAGGTAACAGGAGTTTTCAAGGCCATGGCAGTTCGAGTTGGTCCGAATCAACGAACATTACGAGCATTGTATAAGACCTACATCGATTGCGTGCACGTCAAGAAGTCTGACAGGGGTCGACTGCAAACTGAAGATCCTATGGAGATGGATAAGGAGAATGATATGTATGCTGGGTATCATGAAAGTGATACTTCAGAAGCTGCTAATGAAGCAAAGATTCAAAAACTTAAAGAGCTGTCCAAGCTCCCGGACATTTATGATAGACTTTCAAGGTCGCTGGCTCCAAGCATTTGGGAGCTTGAAGATATTAAAAAGGGTCTTCTTTGCCAGCTCTTTGGTGGGAAGGCTAAGAAAATTCCATCTGGAGCATCTTTCCGAGGTGACATCAATGTTTTACTTGTTGGGGACCCTGGTACCAGTAAATCTCAGCTGCTTCAGTATGTGCACAAGATAGCTCCTCGTGGAATCTACACTAGTGGGCGAGGAAGTTCGGCGGTTGGGCTGACAGCGTATGTAAACGAAGGATCCAGAAACTCGAGAGACGGTATTGGAGAGCGGAGCTTTGGTTCTTAGTGATCGTGGGATATGCTGTATCGATGAGTTCGACAAAATGTCTGATAATGCCCGAAGCATGCTTCATGAGGTAATGGAGCAACAAACGGTATCTGGACCCAAGCGGTTCATGCTCGTGAAG CCGAGTTG

[0213] Nucleotide sequence of the partial CC-3 from Physcomitrellapatens (SEQ ID NO:3)GCACCAGCCGCTTTGGAATCCCATCCCTCGGTTGCATAGACACAAGGGGATTCAGTGTAGTGATACGTTGTGCATATTTGGTGTTGCAAGATTTTTGGTTTCTTGATTGTTAGCTATGGCGTCTGCAACAGCGGCTACAATGGCGTCCCTCCTCACGCCTGGGTCTCTCCGACGCGGTTTGGGTAGCCAGGAATCGTCGACCCAATTTGCTCCCCTAGCTGGTCCTCGTAAGACATCAGTTTCGCGTAGGGTGACTGCTAGCGCTAGTGGGAAGAACGACAATGGAGTCGTGGAAGATGTGGATATGGGGAAGCGGGGTATGTTGAAAGGCGTAGCGGGAGCTTTGGCTGCAGTTCTCCCTGCTGTTATCGCGAAGAAAGCTTCAGCAGCTGAGGAGCAGGGCGTAGCGTCTTCCAGGATGTCCTACTCGAGGTTTTTGGAGTATTTGGATATGGACCGTGTGAAGAAGTTGACTTGTATGAAAATGGGACCATAGCAATTGTGGAGGCTGTATCCCCTGAATTGGGCAACAGAGTGCAACGCGTACGCGTGCAGCTCCCCGGAAC

[0214] Nucleotide sequence of the full-length CC-1 from Physcomitrellapatens (SEQ ID NO:4)GCGATATCGACCCAAGGTGTGTAGAGAAGGGGATAGCCATGTACAGCAACTTCAAAGAGCAGGCCATAGAATATGTGCGTCAAGCCGTAGCGGAAGACAACGCAGGGAACTATGCCAAAGCGTTTCCGCTGTACATGAACGCGCTTGAGTACTTCAAGACGCATCTAAAGTATGAGAAGAATCCCAAAATCAAGGAGGCCATCACTCAGAAGTTCACGGAGTATTTGAGGAGGGCGGAGGAGATTCGAGCCGTTTTGGACGATGGCCCCACTGGACCCTCTGCAAATGGAGACGCGGCAGTTCAAGCTAAACCGAAGTCGAAATCAGGGAAGAAGGATGGTGGCGGGGGTGATGGTGATGGTGACAGCGAGGATCCCGAGCAGCAGAAGCTGAGATCAGGGCTGAACTCGGCAATCATACGGGAAAAGCCAAATGTTCGGTGGGCTGATGTTGCTGGACTTGAAAGTGCCAAGCAGGCGTTGCAGGAGGCAGTGATCTTGCCCGTGAAGTTTCCCCAATTTTTCACAGGGAAGCGAAGACCATGGCGAGCATTTTTGTTGTATGGGCCCCCCGGGACTGGAAAATCGTATCTTGCAAAAGCTGTTGCTACGGAAGCTGATTCTACATTCTTTAGTATTTCCTCTTCAGACTTGGTGTCAAAGTGGATGGGAGAGAGTGAGAAGCTTGTTGCAAATCTGTTTCAAATGGCCCGTGAAGCTGCTCCATCCATCATCTTCATAGACGAGATTGATTCTTTATGCGGTACTCGAGGTGAAGGAAATGAGAGCGAGGCTTCACGTCGTATCAAGACTGAGTTGCTAGTTCAAATGCAGGGTGTCGGCAATCAAGACACTAAGGTTCTTGTGTTAGCTGCTACAAATACGCCCTACTCTTTGGATCAGGCGGTGAGGCGACGTTTCGACAAGCGTATCTACATCCCACTACCGGAGTCTAAGGCTCGGCAGCACATGTTTAAGGTGCATTTGGGAGATACGCCAAACAACCTGACTGAACGTGATTATGAGGATCTGGCTAGGAAGACTGATGGGTTTTCAGGCTCGGATATTGCAGTTTGTGTGAAAGATGTACTATTTGAGCCTGTTCGTAAGACCCAAGATGCTATGCATTTCAAAAGAATTAATACCAAAGAAGGAGAGATGTGGATGCCTTGTGGGCCCCGAGAACCAGGTGCTAGGCAAACCACTATGACGGAGCTTGCCGCTGAAGGGCAGGCATCGAAGATTTTACCACCTCCAATCACAAAATCAGATTTCGACAAAGTCCTCGCAAAGCAGAGGCCCACTGTCAGCAAAGGCGACCTTATTATTCAAGAGAAATTCACCAAAGAATTTGGTGAAGAAGGTTGAATGGTGCTTCGAGTTAAGAATTTGGAAGGTTCTGGGTTACGGAAGACAGACGAAATAGAACGTCGTAGGTACGGTAGCCTAAGAGTAAATTACGGAAGTTTTCCGACTTGCCAGTTGTGCACTCTTTCAACATGAAGGGAAGGAAGCTACTTGTCGGTTGCCTTTTCATGGGTGAGTTGCATCAGTCGCGAGCTCGC

[0215] Nucleotide sequence of the full-length CC-2 from Physcomitrellapatens (SEQ ID NO:5)ATGGCGCGCCGCACTCACGTGAGGAATTGCACCTCCTTGTTCTGCGACGGTTCCATTCTTTTTGGTTTTTAGTTTGCAAATCTTGATCGTGGAGTTGAGAAAAAGGGCGGTTCGTTGTCTTGAGGTGTTCTTGTTGATTGTTCGTCATGGAAAATAATGATGCACTTGACATTGGAGCCGTGTCGTCCCCATATCCTTCGCAATCTGAAGGAGTGTCTACGCCATTGCCGCAAGTAACATCACCGAGCTTCGACAATGCAGCCTCACCCGTGGCCGGGCGGAGGGCCGTACGGCAGACCCCTACATCTGCAGTTCGAAGGAGAGGGAGAGAAACGGATTCCGCTCGTCGTAGGAGGAGTCGATCTCGCAGTTTAGGCAATTCTGTTTATAGTTCCCCTTACGATGCGGGGACTCCTGGAACTCCTGGAACTCCAGTGGCTACTCCGGTTTACGCTACCCCAGTCGGTACACCTATGGGTACCCCATCGTTCCATCGTGGCACGCCACAGTACAAACAGCGCAGTGAGCTTGGTTCCCAGGGGAAGCCTCTACATCGGAGACGTCGATCTCAATCCAGAGAACCCGGGCATCGATCTCCTTCAAGGGAACCTAGTGCTGATGGGCGTCCCTCTGAATCTGCTGAGCCAGATGACACTTTGGGTGGAGAATATGCTTATGTTTGGGGGACGAATGTTAACATTCCAGATGTGCTTAGGGCGATTCGTCGATTTCTCCACAATTATCGTTCGAGTGCTCATGATCTTAATTCCAAGTACATCCAGATCATAGAGGAGACTGTGGAGCGTGAGGAGGATACTCTAAATATCGACATGTCAGACATTTATGACCATGATCCTGATCTATACGCAAAAATTGTTCGATACCCACTCGACATCATCCCCCTGTTGGACACTGAGTGTCAGGAAGTTGCTACCTCTTTACTACCAACGTTTGAGAAGCATATTGAGGCCAGACCTTTCAATCTCAAAGCATCGGTGCACATGCGTGAACTCAACCCTTCAGATATAGACAAATTGGTTTCTGTTAAAGGAATGGTTATCCGGTGCAGTTCTATCATACCTGAAATTAAGGGGGCCTTCTTCAAATGTTTAGTGTGTGGTCACTCGCCTCCGCTAGTTACAGTTGTTAAAGGGCGGGTTGAGGAGCCAACAAGGTGTGAAAAGCCAGAATGTGCAGCACGGAATGCTATGTCTCTTATTCACAATCGATGCACTTTTGCAAATAAGCAGATAGTGCGTCTTCAAGAAACTCCAGATGCCATTCCTGAAGGAGAGACTCCACACACAGTCAGCATGTGTTTATACAACACTATGGTTGATGCTGTGAAGCCTGGAGATCGTATTGAGGTAACAGGAGTTTTCAAGGCCATGGCAGTTCGAGTTGGTCCGAATCAACGAACATTACGAGCATTGTATAAGACCTACATCGATTGCGTGCACGTCAAGAAGTCTGACAGGGGTCGACTGCAAACTGAAGATCCTATGGAGATGGATAAGGAGAATGATATGTATGCTGGGTATCATGAAAGTGATACTTCAGAAGCTGCTAATGAAGCAAAGATTCAAAAACTTAAAGAGCTGTCCAAGCTCCCGGGCATTTATGATAGACTTTCAAGGTCGCTGGCTCCAAGCATTTGGGAGCTTGAAGATATTAAAAAGGGTCTTCTTTGCCAGCTCTTTGGTGGGAAGGCTAAGAAAATTCCATCTGGAGCATCTTTCCGAGGTGACATCAATGTTTTACTTGTTGGGGACCCTGGTACCAGTAAATCTCAGCTGCTTCAGTATGTGCACAAGATAGCTCCTCGTGGAATCTACACTAGTGGGCGAGGAAGTTCGGCGGTTGGGCTGACAGCGTATGTAACGAAGGATCCAGAAACTCGAGAGACGGTATTGGAGAGCGGAGCTTTGGTTCTTAGTGATCGTGGGATATGCTGTATCGATGAGTTCGACAAAATGTCTGATAATGCCCGAAGCATGCTTCATGAGGTAATGGAGCAACAAACGGTATCTGTAGCCAAAGGGGGTATCATTGCCTCGCTGAACGCTCGGACGTCTGTCCTTGCATGTGCAAATCCTAGTGGGTCCCGATACAATGCGCGCCTTTCTGTGATTGATAACATCCAGCTTCCTCCAACTCTACTTTCTAGATTTGATTTAATTTACTTAATGCTCGACAAACCAGACGAGCAAAACGATCGTCGTCTCGCCAGGCATCTCGTGGCTTTACACTATGAAAACTATGAAGTTTCAAAGCAGGACGCCTTAGATCTACAAACACTTACCGCGTATATCACCTATGCTCGTCAGCATGTACATCCTACATTAAGTGATGAAGCTGCTGAAGATTTGATTAATGGCTATGTTGAGATGCGCCAAAAGGGCAACTTTCCTGGAAGCAGTAAAAAGGTGATAACAGCCACACCTCGGCAACTCGAAAGTATGATTCGTATCAGTGAAGCCCTAGCTCGAATGAGATTTTCTGAAGTGGTAGAGAAAGTTGATGCAGCAGAAGCTGTGCGCCTTTTAGACGTCGCTTTGCAGCAATCTGCTACTGATCATGCAACAGGTACGATAGACATGGATCTTATCACGACTGGAGTGTCGGCCAGCGAGCGTATTCGTCGGGCCAACTTGCTAGCTGCTCTGCGAGAGCTTATAGCAGATAAAATTTCACCTGGCAGCTCCTCTGGCTTGAAGACCAGTCAGCTTCTTGAGGATATCCGGAGCCAAAGCAGTGTGGACGTTAGTTTGCAGGATATTAAAAATGCTCTGGGTAGCCTCCAAGGAGAAGGCTTTCTTACTGTCCATGGTGACATAGTCAAGAGAGTTTGAGACAGTTTCTAACTGTTCGAATCCATGAGCTATAACTCTGAACGAAAGGGAAAACCTCCAGTTTCCCATGCGCAAT TCCCAGAGCTCGC

[0216] Nucleotide sequence of the full-length CC-3 from Physcomitrellapatens (SEQ ID NO:6)ATCCCGGGTGATACGTTGTGCATATTTGGTGTTGCAAGTTTTTTGGTTTCTTGATTGTTAGCTATGGCGTCTGCAACAGCGGCTACAATGGCGTCCCTCCTCACGCCTGGGTCTCTCCGACGCGGTTTGGGTAGCCAGGAATCGTCGACCCAATTTGCTCCCCTAGCTGGTCCTCGTAAGACATCAGTTTCGCGTAGGGTGACTGCTAGCGCTAGTGGGAAGAACGACAATGGAGTCGTGGAAGATGTGGATATGGGGAAGCGGGGTATGTTGAAAGGCGTAGCGGGAGCTTTGGCTGCAGTTCTCCCTGCTGTTATCGCGAAGAAAGCTTCAGCAGCTGAGGAGCAGGGCGTAGCGTCTTCCAGGATGTCCTACTCGAGGTTTTTGGAGTATTTGGATATGGACCGTGTGAAGAAGGTTGACTTGTATGAAAATGGGACCATAGCAATTGTGGAGGCTGTATCCCCTGAATTGGGCAACAGAGTACAACGCGTACGCGTGCAGCTCCCCGGAACTAGCTCCGAATTGTTGTCGAAGTTTAGATCGAAGAATGTAGATTTTGCCGCACACAGCCCACAAGAGGACTCTGGCTCTGTCATTTTGAACCTCATCGGAAATTTGGCTTTCCCCTTGTTGCTCGTTGGAGGTCTGTTCTTCTTGTCTCGTAGATCCCAAGGTGGTATGGGACCTGGCGGTCCTGGGAACCCGATGGCCTTCGGGAAGTCCAAGGCCAAGTTCCAGATGGAGCCCAACACGGGCATTACATTCCAAGATGTTGCGGGAGTAGACGAGGCCAAGCAAGACTTCATGGAGGTTGTGGAGTTCTTGAAGCGGCCTGAGAGATTCACAGCAGTGGGCGCTAAAATCCCAAAGGGAGTGTTGCTGGTTGGACCACCCGGTACCGGAAAGACTCTATTGGCGAAGGCCATCGCTGGGGAAGCTGGAGTACCATTCTTCTCCATCTCTGGGTCTGAGTTCGTGGAAATGTTCGTGGGAGTGGGAGCTTCCCGTGTGAGGGACTTGTTCAAGAAGGCGAAAGAGAATGCTCCCTGCATTGTGTTTGTGGATGAGATTGATGCCGTTGGAAGACAGAGAGGAACTGGAATTGGAGGAGGCAATGATGAGCGTGAGCAGACGTTGAATCAGTTGTTGACGGAGATGGACGGTTTCGAAGGAAACACTGGTGTGATTGTCATTGCTGCCACCAACAGGGCTGATATTCTCGACGCTGCCTTGCTTCGTCCTGGAAGATTCGACAGACAGGTTTCCGTGGATGTTCCGGACGTGAAGGGAAGGACTGACATCCTCAAGGTGCATGCTAGTAACAAGAAGTTCGCCGATGATGTGTCTCTGGATATCATTGCCATGAGGACACCAGGGTTCAGTGGAGCTGACTTGGCCAACTTGTTGAACGAAGCAGCTATCTTGACCGGAAGGAGAGGAAAGACAGCCATCAGTGCTAAGGAGATTGACGATTCAATTGACAGAATCGTTGCCGGGATGGAAGGTACCGTCATGACGGACGGCAAGAGCAAGAGTCTTGTTGCATACCACGAAGTCGGCCATGCTATCTGCGGGACTTTGACTCCCGGACACGACGCCGTGCAAAAGGTCACCCTCATTCCTAGAGGTCAGGCCCGTGGTCTCACCTGGTTTATTCCCGGAGAAGACCCGACTTTGATTTCAAAGCAGCAAATCTTTGCCCGTATTGTTGGTGCTCTTGGTGGTAGGGCTACCGAGCAGGTTGTCTTCGGTGATGCTGAAGTTACGACTGGAGCGTCCAGTGATTTGCAGCAAGTTACTTCTATGGCCAAGCAGATGGTCACAGTATTCGGTATGTCAGACATCGGCCCATGGGCTTTGATGGACCCTTCATCCCAGGGAGGAGATATGATTATGCGTATGATGGCACGTAACTCCATGTCCGAGAAGTTGGCTGAGGACATCGACAAGGCTGTGAAGGCTATCTCTGACGAGGCCTACGAAGTCGCACTGGGTCACATTAGGAACAACCGCACGGCCATGGACAAGATTGTAGAGGTTCTGCTTGAGAAGGAGACTTTGTCCGGCGCCGAGTTTAGGGCTATTCTTTCGGAATACACAGAGATTCCTGCTGAAAACCGTGTATCAGACAACCAGGCCGCACCTGTAGCTGTTTGAAGTGATCAGAAGCAAGGGTGTTTGTGAACACAATCGTGTAGGTTTTGGAGCATCGACGCTCTTGTATCAGAGCTCGC

[0217] Deduced amino acid sequence of CC-1 from Physcomitrella patens(SEQ ID NO:7) MYSNFKEQAIEYVRQAVAEDNAGNYAKAFPLYMNALEYFKTHLKYEKNPKIKEAITQKFTEYLRRAEEIRAVLDDGPTGPSANGDAAVQAKPKSKSGKKDGGGGDGDGDSEDPEQQKLRSGLNSAIIREKPNVRWADVAGLESAKQALQEAVILPVKFPQFFTGKRRPWRAFLLYGPPGTGKSYLAKAVATEADSTFFSISSSDLVSKWMGESEKLVANLFQMAREAAPSIIFIDEIDSLCGTRGEGNESEASRRIKTELLVQMQGVGNQDTKVLVLAATNTPYSLDQAVRRRFDKRIYIPLPESKARQHMFKVHLGDTPNNLTERDYEDLARKTDGFSGSDIAVCVKDVLFEPVRKTQDAMHFKRINTKEGEMWMPCGPREPGARQTTMTELAAEGQASKILPPPITKSDFDKVLAKQRPTVSKGDLIIQEKFTKEFGEEG

[0218] Deduced amino acid sequence of CC-2 from Physcomitrella patens(SEQ ID NO:8) MENNDALDIGAVSSPYPSQSEGVSTPLPQVTSPSFDNAASPVAGRRAVRQTPTSAVRRRGRETDSARRRRSRSRSLGNSVYSSPYDAGTPGTPGTPVATPVYATPVGTPMGTPSFHRGTPQYKQRSELGSQGKPLHRRRRSQSREPGHRSPSREPSADGRPSESAEPDDTLGGEYAYVWGTNVNIPDVLRAIRRFLHNYRSSAHDLNSKYIQIIEETVEREEDTLNIDMSDIYDHDPDLYAKIVRYPLDIIPLLDTECQEVATSLLPTFEKHIEARPFNLKASVHMRELNPSDIDKLVSVKGMVIRCSSIPEIKGAFFKCLVCGHSPPLVTVVKGRVEEPTRCEKPECAARNAMSLIHNRCTFANKQIVRLQETPDAIPEGETPHTVSMCLYNTMVDAVKPGDRIEVTGVFKAMAVRVGPNQRTLRALYKTYIDCVHVKKSDRGRLQTEDPMEMDKENDMYAGYHESDTSEAANEAKIQKLKELSKLPGIYDRLSRSLAPSIWELEDIKKGLLCQLFGGKAKKIPSGASFRGDINVLLVGDPGTSKSQLLQYVHKIAPRGIYTSGRGSSAVGLTAYVTKDPETRETVLESGALVLSDRGICCIDEFDKMSDNARSMLHEVMEQQTVSVAKGGIIASLNARTSVLACANPSGSRYNARLSVIDNIQLPPTLLSRFDLIYLMLDKPDEQNDRRLARHLVALHYENYEVSKQDALDLQTLTAYITYARQHVHPTLSDEAAEDLINGYVEMRQKGNFPGSSKKVITATPRQLESMIRISEALARMRFSEVVEKVDAAEAVRLLDVALQQSATDHATGTIDMDLITTGVSASERIRRANLLAALRELIADKISPGSSSGLKTSQLLEDIRSQSSVDVSLQDIKNALGSLQGEGFLTVHGDIVKRV

[0219] Deduced amino acid sequence of CC-3 from Physcomitrella patens(SEQ ID NO:9) MASATAATMASLLTPGSLRRGLGSQESSTQFAPLAGPRKTSVSRRVTASASGKNDNGVVEDVDMGKRGMLKGVAGALAAVLPAVIAKKASAAEEQGVASSRMSYSRFLEYLDMDRVKKVDLYENGTIAIVEAVSPELGNRVQRVRVQLPGTSSELLSKFRSKNVDFAAHSPQEDSGSVELNLIGNLAFPLLLVGGLFFLSRRSQGGMGPGGPGNPMAFGKSKAKFQMEPNTGITFQDVAGVDEAKQDFMEVVEFLKRPERFTAVGAKJPKGVLLVGPPGTGKTLLAKAIAGEAGVPFFSISGSEFVEMFVGVGASRVRDLFKKAKENAPCIVFVDEIDAVGRQRGTGIGGGNDEREQTLNQLLTEMDGFEGNTGVIVIAATNRADELDAALLRPGRFDRQVSVDVPDVKGRTDILKVHASNKKFADDVSLDIIAMRTPGFSGADLANLLNEAAILTGRRGKTAISAKEIDDSIDRIVAGMEGTVMTDGKSKSLVAYHEVGHAICGTLTPGHDAVQKVTLIPRGQARGLTWFIPGEDPTLISKQQIFARIVGALGGRATEQVVFGDAEVTTGASSDLQQVTSMAKQMVTVFGMSDIGPWALMDPSSQGGDMIMRMMARNSMSEKLAEDIDKAVKAISDEAYEVALGHIRNNRTAMDKIVEVLLEKETLSGAEFRAILSEYTEIPAENRVSDNQAAPVAV

1 34 1 887 DNA Physcomitrella patens modified_base (71) a, t, c, g,other or unknown 1 cgggagttgg tgatcttcag catgctcgta gttgacaagggaggtaggat tgtttgaccc 60 aaggtgtgta nagaagggga tagccatgta cagcaacttcaaagagcagg ccatagaata 120 ttgtgcgtca agccgtagcg gaagacaacg cagggaactatgccaaagcg tttccgctgt 180 acatgaacgc gcttgagtac ttcaagacgc atctaaagtatgagaagaat cccaaaatca 240 aggaggccat cactcagaag ttcacggagt atttgaggagggcggaggag attcgagccg 300 ttttggacga tggccccact ggaccctctg caaatggagacgcggcagtt caagctaaac 360 cgaagtcgaa atcagggaag aaggatggtg gcgggggtgatggtgatggt gacagcgagg 420 atcccgacca gcagaagctg agatcagggc tgaactcggcaatcatacgg gaaaagccaa 480 atgttcggtg ggctgatgtt gctggacttg aaagtgccaagcaggcgttg caggaggcag 540 tgatcttgcc cgtgaagttt ccccaatttt tcacagggaagcgaagaaca tggcgagcat 600 ttttgtggta tgggcccccc gggactggaa aatcgtatcttgcaaaagct gttgctacgg 660 aagctgattc tacattcttt agtatttcct cttcagacttggtgtcaaag tggatgggag 720 agagtgagaa gcttgttgca aatctgtttc aaatggcccgtgaagctgct ccatccatca 780 tcttcataga cgagattgat tctttatgcg gtactcgaggtgaaggaaat gagagcgagg 840 cttcacgtcg tatcaagact gagttgctag ttcaaatgcagggtgtc 887 2 723 DNA Physcomitrella patens 2 cggcaccagg gagatcgtattgaggtaaca ggagttttca aggccatggc agttcgagtt 60 ggtccgaatc aacgaacattacgagcattg tataagacct acatcgattg cgtgcacgtc 120 aagaagtctg acaggggtcgactgcaaact gaagatccta tggagatgga taaggagaat 180 gatatgtatg ctgggtatcatgaaagtgat acttcagaag ctgctaatga agcaaagatt 240 caaaaactta aagagctgtccaagctcccg gacatttatg atagactttc aaggtcgctg 300 gctccaagca tttgggagcttgaagatatt aaaaagggtc ttctttgcca gctctttggt 360 gggaaggcta agaaaattccatctggagca tctttccgag gtgacatcaa tgttttactt 420 gttggggacc ctggtaccagtaaatctcag ctgcttcagt atgtgcacaa gatagctcct 480 cgtggaatct acactagtgggcgaggaagt tcggcggttg ggctgacagc gtatgtaaac 540 gaaggatcca gaaactcgagagacggtatt ggagagcgga gctttggttc ttagtgatcg 600 tgggatatgc tgtatcgatgagttcgacaa aatgtctgat aatgcccgaa gcatgcttca 660 tgaggtaatg gagcaacaaacggtatctgg acccaagcgg ttcatgctcg tgaagccgag 720 ttg 723 3 566 DNAPhyscomitrella patens 3 gcaccagccg ctttggaatc ccatccctcg gttgcatagacacaagggga ttcagtgtag 60 tgatacgttg tgcatatttg gtgttgcaag atttttggtttcttgattgt tagctatggc 120 gtctgcaaca gcggctacaa tggcgtccct cctcacgcctgggtctctcc gacgcggttt 180 gggtagccag gaatcgtcga cccaatttgc tcccctagctggtcctcgta agacatcagt 240 ttcgcgtagg gtgactgcta gcgctagtgg gaagaacgacaatggagtcg tggaagatgt 300 ggatatgggg aagcggggta tgttgaaagg cgtagcgggagctttggctg cagttctccc 360 tgctgttatc gcgaagaaag cttcagcagc tgaggagcagggcgtagcgt cttccaggat 420 gtcctactcg aggtttttgg agtatttgga tatggaccgtgtgaagaagt tgacttgtat 480 gaaaatggga ccatagcaat tgtggaggct gtatcccctgaattgggcaa cagagtgcaa 540 cgcgtacgcg tgcagctccc cggaac 566 4 1564 DNAPhyscomitrella patens 4 gcgatatcga cccaaggtgt gtagagaagg ggatagccatgtacagcaac ttcaaagagc 60 aggccataga atatgtgcgt caagccgtag cggaagacaacgcagggaac tatgccaaag 120 cgtttccgct gtacatgaac gcgcttgagt acttcaagacgcatctaaag tatgagaaga 180 atcccaaaat caaggaggcc atcactcaga agttcacggagtatttgagg agggcggagg 240 agattcgagc cgttttggac gatggcccca ctggaccctctgcaaatgga gacgcggcag 300 ttcaagctaa accgaagtcg aaatcaggga agaaggatggtggcgggggt gatggtgatg 360 gtgacagcga ggatcccgag cagcagaagc tgagatcagggctgaactcg gcaatcatac 420 gggaaaagcc aaatgttcgg tgggctgatg ttgctggacttgaaagtgcc aagcaggcgt 480 tgcaggaggc agtgatcttg cccgtgaagt ttccccaatttttcacaggg aagcgaagac 540 catggcgagc atttttgttg tatgggcccc ccgggactggaaaatcgtat cttgcaaaag 600 ctgttgctac ggaagctgat tctacattct ttagtatttcctcttcagac ttggtgtcaa 660 agtggatggg agagagtgag aagcttgttg caaatctgtttcaaatggcc cgtgaagctg 720 ctccatccat catcttcata gacgagattg attctttatgcggtactcga ggtgaaggaa 780 atgagagcga ggcttcacgt cgtatcaaga ctgagttgctagttcaaatg cagggtgtcg 840 gcaatcaaga cactaaggtt cttgtgttag ctgctacaaatacgccctac tctttggatc 900 aggcggtgag gcgacgtttc gacaagcgta tctacatcccactaccggag tctaaggctc 960 ggcagcacat gtttaaggtg catttgggag atacgccaaacaacctgact gaacgtgatt 1020 atgaggatct ggctaggaag actgatgggt tttcaggctcggatattgca gtttgtgtga 1080 aagatgtact atttgagcct gttcgtaaga cccaagatgctatgcatttc aaaagaatta 1140 ataccaaaga aggagagatg tggatgcctt gtgggccccgagaaccaggt gctaggcaaa 1200 ccactatgac ggagcttgcc gctgaagggc aggcatcgaagattttacca cctccaatca 1260 caaaatcaga tttcgacaaa gtcctcgcaa agcagaggcccactgtcagc aaaggcgacc 1320 ttattattca agagaaattc accaaagaat ttggtgaagaaggttgaatg gtgcttcgag 1380 ttaagaattt ggaaggttct gggttacgga agacagacgaaatagaacgt cgtaggtacg 1440 gtagcctaag agtaaattac ggaagttttc cgacttgccagttgtgcact ctttcaacat 1500 gaagggaagg aagctacttg tcggttgcct tttcatgggtgagttgcatc agtcgcgagc 1560 tcgc 1564 5 4348 DNA Physcomitrella patens 5atggcgcgcc gcactcacgt gaggaattgc acctccttgt tctgcgacgg ttccattctt 60tttggttttt agtttgcaaa tcttgatcgt ggagttgaga aaaagggcgg ttcgttgtct 120tgaggtgttc ttgttgattg ttcgtcatgg aaaataatga tgcacttgac attggagccg 180tgtcgtcccc atatccttcg caatctgaag gagtgtctac gccattgccg caagtaacat 240caccgagctt cgacaatgca gcctcacccg tggccgggcg gagggccgta cggcagaccc 300ctacatctgc agttcgaagg agagggagag aaacggattc cgctcgtcgt aggaggagtc 360gatctcgcag tttaggcaat tctgtttata gttcccctta cgatgcgggg actcctggaa 420ctcctggaac tccagtggct actccggttt acgctacccc agtcggtaca cctatgggta 480ccccatcgtt ccatcgtggc acgccacagt acaaacagcg cagtgagctt ggttcccagg 540ggaagcctct acatcggaga cgtcgatctc aatccagaga acccgggcat cgatctcctt 600caagggaacc tagtgctgat gggcgtccct ctgaatctgc tgagccagat gacactttgg 660gtggagaata tgcttatgtt tgggggacga atgttaacat tccagatgtg cttagggcga 720ttcgtcgatt tctccacaat tatcgttcga gtgctcatga tcttaattcc aagtacatcc 780agatcataga ggagactgtg gagcgtgagg aggatactct aaatatcgac atgtcagaca 840tttatgacca tgatcctgat ctatacgcaa aaattgttcg atacccactc gacatcatcc 900ccctgttgga cactgagtgt caggaagttg ctacctcttt actaccaacg tttgagaagc 960atattgaggc cagacctttc aatctcaaag catcggtgca catgcgtgaa ctcaaccctt 1020cagatataga caaattggtt tctgttaaag gaatggttat ccggtgcagt tctatcatac 1080ctgaaattaa gggggccttc ttcaaatgtt tagtgtgtgg tcactcgcct ccgctagtta 1140cagttgttaa agggcgggtt gaggagccaa caaggtgtga aaagccagaa tgtgcagcac 1200ggaatgctat gtctcttatt cacaatcgat gcacttttgc aaataagcag atagtgcgtc 1260ttcaagaaac tccagatgcc attcctgaag gagagactcc acacacagtc agcatgtgtt 1320tatacaacac tatggttgat gctgtgaagc ctggagatcg tattgaggta acaggagttt 1380tcaaggccat ggcagttcga gttggtcatg gcgcgccgca ctcacgtgag gaattgcacc 1440tccttgttct gcgacggttc cattcttttt ggtttttagt ttgcaaatct tgatcgtgga 1500gttgagaaaa agggcggttc gttgtcttga ggtgttcttg ttgattgttc gtcatggaaa 1560ataatgatgc acttgacatt ggagccgtgt cgtccccata tccttcgcaa tctgaaggag 1620tgtctacgcc attgccgcaa gtaacatcac cgagcttcga caatgcagcc tcacccgtgg 1680ccgggcggag ggccgtacgg cagaccccta catctgcagt tcgaaggaga gggagagaaa 1740cggattccgc tcgtcgtagg aggagtcgat ctcgcagttt aggcaattct gtttatagtt 1800ccccttacga tgcggggact cctggaactc ctggaactcc agtggctact ccggtttacg 1860ctaccccagt cggtacacct atgggtaccc catcgttcca tcgtggcacg ccacagtaca 1920aacagcgcag tgagcttggt tcccagggga agcctctaca tcggagacgt cgatctcaat 1980ccagagaacc cgggcatcga tctccttcaa gggaacctag tgctgatggg cgtccctctg 2040aatctgctga gccagatgac actttgggtg gagaatatgc ttatgtttgg gggacgaatg 2100ttaacattcc agatgtgctt agggcgattc gtcgatttct ccacaattat cgttcgagtg 2160ctcatgatct taattccaag tacatccaga tcatagagga gactgtggag cgtgaggagg 2220atactctaaa tatcgacatg tcagacattt atgaccatga tcctgatcta tacgcaaaaa 2280ttgttcgata cccactcgac atcatccccc tgttggacac tgagtgtcag gaagttgcta 2340cctctttact accaacgttt gagaagcata ttgaggccag acctttcaat ctcaaagcat 2400cggtgcacat gcgtgaactc aacccttcag atatagacaa attggtttct gttaaaggaa 2460tggttatccg gtgcagttct atcatacctg aaattaaggg ggccttcttc aaatgtttag 2520tgtgtggtca ctcgcctccg ctagttacag ttgttaaagg gcgggttgag gagccaacaa 2580ggtgtgaaaa gccagaatgt gcagcacgga atgctatgtc tcttattcac aatcgatgca 2640cttttgcaaa taagcagata gtgcgtcttc aagaaactcc agatgccatt cctgaaggag 2700agactccaca cacagtcagc atgtgtttat acaacactat ggttgatgct gtgaagcctg 2760gagatcgtat tgaggtaaca ggagttttca aggccatggc agttcgagtt ggtccgaatc 2820aacgaacatt acgagcattg tataagacct acatcgattg cgtgcacgtc aagaagtctg 2880acaggggtcg actgcaaact gaagatccta tggagatgga taaggagaat gatatgtatg 2940ctgggtatca tgaaagtgat acttcagaag ctgctaatga agcaaagatt caaaaactta 3000aagagctgtc caagctcccg ggcatttatg atagactttc aaggtcgctg gctccaagca 3060tttgggagct tgaagatatt aaaaagggtc ttctttgcca gctctttggt gggaaggcta 3120agaaaattcc atctggagca tctttccgag gtgacatcaa tgttttactt gttggggacc 3180ctggtaccag taaatctcag ctgcttcagt atgtgcacaa gatagctcct cgtggaatct 3240acactagtgg gcgaggaagt tcggcggttg ggctgacagc gtatgtaacg aaggatccag 3300aaactcgaga gacggtattg gagagcggag ctttggttct tagtgatcgt gggatatgct 3360gtatcgatga gttcgacaaa atgtctgata atgcccgaag catgcttcat gaggtaatgg 3420agcaacaaac ggtatctgta gccaaagggg gtatcattgc ctcgctgaac gctcggacgt 3480ctgtccttgc atgtgcaaat cctagtgggt cccgatacaa tgcgcgcctt tctgtgattg 3540ataacatcca gcttcctcca actctacttt ctagatttga tttaatttac ttaatgctcg 3600acaaaccaga cgagcaaaac gatcgtcgtc tcgccaggca tctcgtggct ttacactatg 3660aaaactatga agtttcaaag caggacgcct tagatctaca aacacttacc gcgtatatca 3720cctatgctcg tcagcatgta catcctacat taagtgatga agctgctgaa gatttgatta 3780atggctatgt tgagatgcgc caaaagggca actttcctgg aagcagtaaa aaggtgataa 3840cagccacacc tcggcaactc gaaagtatga ttcgtatcag tgaagcccta gctcgaatga 3900gattttctga agtggtagag aaagttgatg cagcagaagc tgtgcgcctt ttagacgtcg 3960ctttgcagca atctgctact gatcatgcaa caggtacgat agacatggat cttatcacga 4020ctggagtgtc ggccagcgag cgtattcgtc gggccaactt gctagctgct ctgcgagagc 4080ttatagcaga taaaatttca cctggcagct cctctggctt gaagaccagt cagcttcttg 4140aggatatccg gagccaaagc agtgtggacg ttagtttgca ggatattaaa aatgctctgg 4200gtagcctcca aggagaaggc tttcttactg tccatggtga catagtcaag agagtttgag 4260acagtttcta actgttcgaa tccatgagct ataactctga acgaaaggga aaacctccag 4320tttcccatgc gcaattccca gagctcgc 4348 6 2237 DNA Physcomitrella patens 6atcccgggtg atacgttgtg catatttggt gttgcaagtt ttttggtttc ttgattgtta 60gctatggcgt ctgcaacagc ggctacaatg gcgtccctcc tcacgcctgg gtctctccga 120cgcggtttgg gtagccagga atcgtcgacc caatttgctc ccctagctgg tcctcgtaag 180acatcagttt cgcgtagggt gactgctagc gctagtggga agaacgacaa tggagtcgtg 240gaagatgtgg atatggggaa gcggggtatg ttgaaaggcg tagcgggagc tttggctgca 300gttctccctg ctgttatcgc gaagaaagct tcagcagctg aggagcaggg cgtagcgtct 360tccaggatgt cctactcgag gtttttggag tatttggata tggaccgtgt gaagaaggtt 420gacttgtatg aaaatgggac catagcaatt gtggaggctg tatcccctga attgggcaac 480agagtacaac gcgtacgcgt gcagctcccc ggaactagct ccgaattgtt gtcgaagttt 540agatcgaaga atgtagattt tgccgcacac agcccacaag aggactctgg ctctgtcatt 600ttgaacctca tcggaaattt ggctttcccc ttgttgctcg ttggaggtct gttcttcttg 660tctcgtagat cccaaggtgg tatgggacct ggcggtcctg ggaacccgat ggccttcggg 720aagtccaagg ccaagttcca gatggagccc aacacgggca ttacattcca agatgttgcg 780ggagtagacg aggccaagca agacttcatg gaggttgtgg agttcttgaa gcggcctgag 840agattcacag cagtgggcgc taaaatccca aagggagtgt tgctggttgg accacccggt 900accggaaaga ctctattggc gaaggccatc gctggggaag ctggagtacc attcttctcc 960atctctgggt ctgagttcgt ggaaatgttc gtgggagtgg gagcttcccg tgtgagggac 1020ttgttcaaga aggcgaaaga gaatgctccc tgcattgtgt ttgtggatga gattgatgcc 1080gttggaagac agagaggaac tggaattgga ggaggcaatg atgagcgtga gcagacgttg 1140aatcagttgt tgacggagat ggacggtttc gaaggaaaca ctggtgtgat tgtcattgct 1200gccaccaaca gggctgatat tctcgacgct gccttgcttc gtcctggaag attcgacaga 1260caggtttccg tggatgttcc ggacgtgaag ggaaggactg acatcctcaa ggtgcatgct 1320agtaacaaga agttcgccga tgatgtgtct ctggatatca ttgccatgag gacaccaggg 1380ttcagtggag ctgacttggc caacttgttg aacgaagcag ctatcttgac cggaaggaga 1440ggaaagacag ccatcagtgc taaggagatt gacgattcaa ttgacagaat cgttgccggg 1500atggaaggta ccgtcatgac ggacggcaag agcaagagtc ttgttgcata ccacgaagtc 1560ggccatgcta tctgcgggac tttgactccc ggacacgacg ccgtgcaaaa ggtcaccctc 1620attcctagag gtcaggcccg tggtctcacc tggtttattc ccggagaaga cccgactttg 1680atttcaaagc agcaaatctt tgcccgtatt gttggtgctc ttggtggtag ggctaccgag 1740caggttgtct tcggtgatgc tgaagttacg actggagcgt ccagtgattt gcagcaagtt 1800acttctatgg ccaagcagat ggtcacagta ttcggtatgt cagacatcgg cccatgggct 1860ttgatggacc cttcatccca gggaggagat atgattatgc gtatgatggc acgtaactcc 1920atgtccgaga agttggctga ggacatcgac aaggctgtga aggctatctc tgacgaggcc 1980tacgaagtcg cactgggtca cattaggaac aaccgcacgg ccatggacaa gattgtagag 2040gttctgcttg agaaggagac tttgtccggc gccgagttta gggctattct ttcggaatac 2100acagagattc ctgctgaaaa ccgtgtatca gacaaccagg ccgcacctgt agctgtttga 2160agtgatcaga agcaagggtg tttgtgaaca caatcgtgta ggttttggag catcgacgct 2220cttgtatcag agctcgc 2237 7 442 PRT Physcomitrella patens 7 Met Tyr SerAsn Phe Lys Glu Gln Ala Ile Glu Tyr Val Arg Gln Ala 1 5 10 15 Val AlaGlu Asp Asn Ala Gly Asn Tyr Ala Lys Ala Phe Pro Leu Tyr 20 25 30 Met AsnAla Leu Glu Tyr Phe Lys Thr His Leu Lys Tyr Glu Lys Asn 35 40 45 Pro LysIle Lys Glu Ala Ile Thr Gln Lys Phe Thr Glu Tyr Leu Arg 50 55 60 Arg AlaGlu Glu Ile Arg Ala Val Leu Asp Asp Gly Pro Thr Gly Pro 65 70 75 80 SerAla Asn Gly Asp Ala Ala Val Gln Ala Lys Pro Lys Ser Lys Ser 85 90 95 GlyLys Lys Asp Gly Gly Gly Gly Asp Gly Asp Gly Asp Ser Glu Asp 100 105 110Pro Glu Gln Gln Lys Leu Arg Ser Gly Leu Asn Ser Ala Ile Ile Arg 115 120125 Glu Lys Pro Asn Val Arg Trp Ala Asp Val Ala Gly Leu Glu Ser Ala 130135 140 Lys Gln Ala Leu Gln Glu Ala Val Ile Leu Pro Val Lys Phe Pro Gln145 150 155 160 Phe Phe Thr Gly Lys Arg Arg Pro Trp Arg Ala Phe Leu LeuTyr Gly 165 170 175 Pro Pro Gly Thr Gly Lys Ser Tyr Leu Ala Lys Ala ValAla Thr Glu 180 185 190 Ala Asp Ser Thr Phe Phe Ser Ile Ser Ser Ser AspLeu Val Ser Lys 195 200 205 Trp Met Gly Glu Ser Glu Lys Leu Val Ala AsnLeu Phe Gln Met Ala 210 215 220 Arg Glu Ala Ala Pro Ser Ile Ile Phe IleAsp Glu Ile Asp Ser Leu 225 230 235 240 Cys Gly Thr Arg Gly Glu Gly AsnGlu Ser Glu Ala Ser Arg Arg Ile 245 250 255 Lys Thr Glu Leu Leu Val GlnMet Gln Gly Val Gly Asn Gln Asp Thr 260 265 270 Lys Val Leu Val Leu AlaAla Thr Asn Thr Pro Tyr Ser Leu Asp Gln 275 280 285 Ala Val Arg Arg ArgPhe Asp Lys Arg Ile Tyr Ile Pro Leu Pro Glu 290 295 300 Ser Lys Ala ArgGln His Met Phe Lys Val His Leu Gly Asp Thr Pro 305 310 315 320 Asn AsnLeu Thr Glu Arg Asp Tyr Glu Asp Leu Ala Arg Lys Thr Asp 325 330 335 GlyPhe Ser Gly Ser Asp Ile Ala Val Cys Val Lys Asp Val Leu Phe 340 345 350Glu Pro Val Arg Lys Thr Gln Asp Ala Met His Phe Lys Arg Ile Asn 355 360365 Thr Lys Glu Gly Glu Met Trp Met Pro Cys Gly Pro Arg Glu Pro Gly 370375 380 Ala Arg Gln Thr Thr Met Thr Glu Leu Ala Ala Glu Gly Gln Ala Ser385 390 395 400 Lys Ile Leu Pro Pro Pro Ile Thr Lys Ser Asp Phe Asp LysVal Leu 405 410 415 Ala Lys Gln Arg Pro Thr Val Ser Lys Gly Asp Leu IleIle Gln Glu 420 425 430 Lys Phe Thr Lys Glu Phe Gly Glu Glu Gly 435 4408 901 PRT Physcomitrella patens 8 Met Glu Asn Asn Asp Ala Leu Asp IleGly Ala Val Ser Ser Pro Tyr 1 5 10 15 Pro Ser Gln Ser Glu Gly Val SerThr Pro Leu Pro Gln Val Thr Ser 20 25 30 Pro Ser Phe Asp Asn Ala Ala SerPro Val Ala Gly Arg Arg Ala Val 35 40 45 Arg Gln Thr Pro Thr Ser Ala ValArg Arg Arg Gly Arg Glu Thr Asp 50 55 60 Ser Ala Arg Arg Arg Arg Ser ArgSer Arg Ser Leu Gly Asn Ser Val 65 70 75 80 Tyr Ser Ser Pro Tyr Asp AlaGly Thr Pro Gly Thr Pro Gly Thr Pro 85 90 95 Val Ala Thr Pro Val Tyr AlaThr Pro Val Gly Thr Pro Met Gly Thr 100 105 110 Pro Ser Phe His Arg GlyThr Pro Gln Tyr Lys Gln Arg Ser Glu Leu 115 120 125 Gly Ser Gln Gly LysPro Leu His Arg Arg Arg Arg Ser Gln Ser Arg 130 135 140 Glu Pro Gly HisArg Ser Pro Ser Arg Glu Pro Ser Ala Asp Gly Arg 145 150 155 160 Pro SerGlu Ser Ala Glu Pro Asp Asp Thr Leu Gly Gly Glu Tyr Ala 165 170 175 TyrVal Trp Gly Thr Asn Val Asn Ile Pro Asp Val Leu Arg Ala Ile 180 185 190Arg Arg Phe Leu His Asn Tyr Arg Ser Ser Ala His Asp Leu Asn Ser 195 200205 Lys Tyr Ile Gln Ile Ile Glu Glu Thr Val Glu Arg Glu Glu Asp Thr 210215 220 Leu Asn Ile Asp Met Ser Asp Ile Tyr Asp His Asp Pro Asp Leu Tyr225 230 235 240 Ala Lys Ile Val Arg Tyr Pro Leu Asp Ile Ile Pro Leu LeuAsp Thr 245 250 255 Glu Cys Gln Glu Val Ala Thr Ser Leu Leu Pro Thr PheGlu Lys His 260 265 270 Ile Glu Ala Arg Pro Phe Asn Leu Lys Ala Ser ValHis Met Arg Glu 275 280 285 Leu Asn Pro Ser Asp Ile Asp Lys Leu Val SerVal Lys Gly Met Val 290 295 300 Ile Arg Cys Ser Ser Ile Ile Pro Glu IleLys Gly Ala Phe Phe Lys 305 310 315 320 Cys Leu Val Cys Gly His Ser ProPro Leu Val Thr Val Val Lys Gly 325 330 335 Arg Val Glu Glu Pro Thr ArgCys Glu Lys Pro Glu Cys Ala Ala Arg 340 345 350 Asn Ala Met Ser Leu IleHis Asn Arg Cys Thr Phe Ala Asn Lys Gln 355 360 365 Ile Val Arg Leu GlnGlu Thr Pro Asp Ala Ile Pro Glu Gly Glu Thr 370 375 380 Pro His Thr ValSer Met Cys Leu Tyr Asn Thr Met Val Asp Ala Val 385 390 395 400 Lys ProGly Asp Arg Ile Glu Val Thr Gly Val Phe Lys Ala Met Ala 405 410 415 ValArg Val Gly Pro Asn Gln Arg Thr Leu Arg Ala Leu Tyr Lys Thr 420 425 430Tyr Ile Asp Cys Val His Val Lys Lys Ser Asp Arg Gly Arg Leu Gln 435 440445 Thr Glu Asp Pro Met Glu Met Asp Lys Glu Asn Asp Met Tyr Ala Gly 450455 460 Tyr His Glu Ser Asp Thr Ser Glu Ala Ala Asn Glu Ala Lys Ile Gln465 470 475 480 Lys Leu Lys Glu Leu Ser Lys Leu Pro Gly Ile Tyr Asp ArgLeu Ser 485 490 495 Arg Ser Leu Ala Pro Ser Ile Trp Glu Leu Glu Asp IleLys Lys Gly 500 505 510 Leu Leu Cys Gln Leu Phe Gly Gly Lys Ala Lys LysIle Pro Ser Gly 515 520 525 Ala Ser Phe Arg Gly Asp Ile Asn Val Leu LeuVal Gly Asp Pro Gly 530 535 540 Thr Ser Lys Ser Gln Leu Leu Gln Tyr ValHis Lys Ile Ala Pro Arg 545 550 555 560 Gly Ile Tyr Thr Ser Gly Arg GlySer Ser Ala Val Gly Leu Thr Ala 565 570 575 Tyr Val Thr Lys Asp Pro GluThr Arg Glu Thr Val Leu Glu Ser Gly 580 585 590 Ala Leu Val Leu Ser AspArg Gly Ile Cys Cys Ile Asp Glu Phe Asp 595 600 605 Lys Met Ser Asp AsnAla Arg Ser Met Leu His Glu Val Met Glu Gln 610 615 620 Gln Thr Val SerVal Ala Lys Gly Gly Ile Ile Ala Ser Leu Asn Ala 625 630 635 640 Arg ThrSer Val Leu Ala Cys Ala Asn Pro Ser Gly Ser Arg Tyr Asn 645 650 655 AlaArg Leu Ser Val Ile Asp Asn Ile Gln Leu Pro Pro Thr Leu Leu 660 665 670Ser Arg Phe Asp Leu Ile Tyr Leu Met Leu Asp Lys Pro Asp Glu Gln 675 680685 Asn Asp Arg Arg Leu Ala Arg His Leu Val Ala Leu His Tyr Glu Asn 690695 700 Tyr Glu Val Ser Lys Gln Asp Ala Leu Asp Leu Gln Thr Leu Thr Ala705 710 715 720 Tyr Ile Thr Tyr Ala Arg Gln His Val His Pro Thr Leu SerAsp Glu 725 730 735 Ala Ala Glu Asp Leu Ile Asn Gly Tyr Val Glu Met ArgGln Lys Gly 740 745 750 Asn Phe Pro Gly Ser Ser Lys Lys Val Ile Thr AlaThr Pro Arg Gln 755 760 765 Leu Glu Ser Met Ile Arg Ile Ser Glu Ala LeuAla Arg Met Arg Phe 770 775 780 Ser Glu Val Val Glu Lys Val Asp Ala AlaGlu Ala Val Arg Leu Leu 785 790 795 800 Asp Val Ala Leu Gln Gln Ser AlaThr Asp His Ala Thr Gly Thr Ile 805 810 815 Asp Met Asp Leu Ile Thr ThrGly Val Ser Ala Ser Glu Arg Ile Arg 820 825 830 Arg Ala Asn Leu Leu AlaAla Leu Arg Glu Leu Ile Ala Asp Lys Ile 835 840 845 Ser Pro Gly Ser SerSer Gly Leu Lys Thr Ser Gln Leu Leu Glu Asp 850 855 860 Ile Arg Ser GlnSer Ser Val Asp Val Ser Leu Gln Asp Ile Lys Asn 865 870 875 880 Ala LeuGly Ser Leu Gln Gly Glu Gly Phe Leu Thr Val His Gly Asp 885 890 895 IleVal Lys Arg Val 900 9 698 PRT Physcomitrella patens 9 Met Ala Ser AlaThr Ala Ala Thr Met Ala Ser Leu Leu Thr Pro Gly 1 5 10 15 Ser Leu ArgArg Gly Leu Gly Ser Gln Glu Ser Ser Thr Gln Phe Ala 20 25 30 Pro Leu AlaGly Pro Arg Lys Thr Ser Val Ser Arg Arg Val Thr Ala 35 40 45 Ser Ala SerGly Lys Asn Asp Asn Gly Val Val Glu Asp Val Asp Met 50 55 60 Gly Lys ArgGly Met Leu Lys Gly Val Ala Gly Ala Leu Ala Ala Val 65 70 75 80 Leu ProAla Val Ile Ala Lys Lys Ala Ser Ala Ala Glu Glu Gln Gly 85 90 95 Val AlaSer Ser Arg Met Ser Tyr Ser Arg Phe Leu Glu Tyr Leu Asp 100 105 110 MetAsp Arg Val Lys Lys Val Asp Leu Tyr Glu Asn Gly Thr Ile Ala 115 120 125Ile Val Glu Ala Val Ser Pro Glu Leu Gly Asn Arg Val Gln Arg Val 130 135140 Arg Val Gln Leu Pro Gly Thr Ser Ser Glu Leu Leu Ser Lys Phe Arg 145150 155 160 Ser Lys Asn Val Asp Phe Ala Ala His Ser Pro Gln Glu Asp SerGly 165 170 175 Ser Val Ile Leu Asn Leu Ile Gly Asn Leu Ala Phe Pro LeuLeu Leu 180 185 190 Val Gly Gly Leu Phe Phe Leu Ser Arg Arg Ser Gln GlyGly Met Gly 195 200 205 Pro Gly Gly Pro Gly Asn Pro Met Ala Phe Gly LysSer Lys Ala Lys 210 215 220 Phe Gln Met Glu Pro Asn Thr Gly Ile Thr PheGln Asp Val Ala Gly 225 230 235 240 Val Asp Glu Ala Lys Gln Asp Phe MetGlu Val Val Glu Phe Leu Lys 245 250 255 Arg Pro Glu Arg Phe Thr Ala ValGly Ala Lys Ile Pro Lys Gly Val 260 265 270 Leu Leu Val Gly Pro Pro GlyThr Gly Lys Thr Leu Leu Ala Lys Ala 275 280 285 Ile Ala Gly Glu Ala GlyVal Pro Phe Phe Ser Ile Ser Gly Ser Glu 290 295 300 Phe Val Glu Met PheVal Gly Val Gly Ala Ser Arg Val Arg Asp Leu 305 310 315 320 Phe Lys LysAla Lys Glu Asn Ala Pro Cys Ile Val Phe Val Asp Glu 325 330 335 Ile AspAla Val Gly Arg Gln Arg Gly Thr Gly Ile Gly Gly Gly Asn 340 345 350 AspGlu Arg Glu Gln Thr Leu Asn Gln Leu Leu Thr Glu Met Asp Gly 355 360 365Phe Glu Gly Asn Thr Gly Val Ile Val Ile Ala Ala Thr Asn Arg Ala 370 375380 Asp Ile Leu Asp Ala Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg Gln 385390 395 400 Val Ser Val Asp Val Pro Asp Val Lys Gly Arg Thr Asp Ile LeuLys 405 410 415 Val His Ala Ser Asn Lys Lys Phe Ala Asp Asp Val Ser LeuAsp Ile 420 425 430 Ile Ala Met Arg Thr Pro Gly Phe Ser Gly Ala Asp LeuAla Asn Leu 435 440 445 Leu Asn Glu Ala Ala Ile Leu Thr Gly Arg Arg GlyLys Thr Ala Ile 450 455 460 Ser Ala Lys Glu Ile Asp Asp Ser Ile Asp ArgIle Val Ala Gly Met 465 470 475 480 Glu Gly Thr Val Met Thr Asp Gly LysSer Lys Ser Leu Val Ala Tyr 485 490 495 His Glu Val Gly His Ala Ile CysGly Thr Leu Thr Pro Gly His Asp 500 505 510 Ala Val Gln Lys Val Thr LeuIle Pro Arg Gly Gln Ala Arg Gly Leu 515 520 525 Thr Trp Phe Ile Pro GlyGlu Asp Pro Thr Leu Ile Ser Lys Gln Gln 530 535 540 Ile Phe Ala Arg IleVal Gly Ala Leu Gly Gly Arg Ala Thr Glu Gln 545 550 555 560 Val Val PheGly Asp Ala Glu Val Thr Thr Gly Ala Ser Ser Asp Leu 565 570 575 Gln GlnVal Thr Ser Met Ala Lys Gln Met Val Thr Val Phe Gly Met 580 585 590 SerAsp Ile Gly Pro Trp Ala Leu Met Asp Pro Ser Ser Gln Gly Gly 595 600 605Asp Met Ile Met Arg Met Met Ala Arg Asn Ser Met Ser Glu Lys Leu 610 615620 Ala Glu Asp Ile Asp Lys Ala Val Lys Ala Ile Ser Asp Glu Ala Tyr 625630 635 640 Glu Val Ala Leu Gly His Ile Arg Asn Asn Arg Thr Ala Met AspLys 645 650 655 Ile Val Glu Val Leu Leu Glu Lys Glu Thr Leu Ser Gly AlaGlu Phe 660 665 670 Arg Ala Ile Leu Ser Glu Tyr Thr Glu Ile Pro Ala GluAsn Arg Val 675 680 685 Ser Asp Asn Gln Ala Ala Pro Val Ala Val 690 69510 18 DNA Artificial Sequence Description of Artificial Sequence Primer10 caggaaacag ctatgacc 18 11 19 DNA Artificial Sequence Description ofArtificial Sequence Primer 11 ctaaagggaa caaaagctg 19 12 18 DNAArtificial Sequence Description of Artificial Sequence Primer 12tgtaaaacga cggccagt 18 13 34 DNA Artificial Sequence Description ofArtificial Sequence Primer 13 gcgatatcga cccaaggtgt gtagagaagg ggat 3414 33 DNA Artificial Sequence Description of Artificial Sequence Primer14 gcgagctcgc gactgatgca actcacccat gaa 33 15 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 15 cccaaatgct tggagccagc gacct25 16 25 DNA Artificial Sequence Description of Artificial SequencePrimer 16 gacgtgcacg caatcgatgt aggtc 25 17 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 17 agtcgacccc tgtcagactt cttga25 18 36 DNA Artificial Sequence Description of Artificial SequencePrimer 18 atggcgcgcc gcactcacgt gaggaattgc acctcc 36 19 32 DNAArtificial Sequence Description of Artificial Sequence Primer 19gcgagctctg ggaattgcgc atgggaaact gg 32 20 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 20 ctggctaccc aaaccgcgtc ggaga25 21 35 DNA Artificial Sequence Description of Artificial SequencePrimer 21 atcccgggtg atacgttgtg catatttggt gttgc 35 22 32 DNA ArtificialSequence Description of Artificial Sequence Primer 22 gcgagctctgatacaagagc gtcgatgctc ca 32 23 30 DNA Artificial Sequence Description ofArtificial Sequence Primer 23 gcgctgcaga tttcatttgg agaggacacg 30 24 35DNA Artificial Sequence Description of Artificial Sequence Primer 24cgcggccggc ctcagaagaa ctcgtcaaga aggcg 35 25 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 25 gctgacacgc caagcctcgc tagtc25 26 33 DNA Artificial Sequence Description of Artificial SequencePrimer 26 gcgagctcgc gactgatgca actcacccat gaa 33 27 32 DNA ArtificialSequence Description of Artificial Sequence Primer 27 gcgagctctgggaattgcgc atgggaaact gg 32 28 32 DNA Artificial Sequence Description ofArtificial Sequence Primer 28 gcgagctctg atacaagagc gtcgatgctc ca 32 2925 DNA Artificial Sequence Description of Artificial Sequence Primer 29gatggtgatg gtgacagcga ggatc 25 30 26 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 30 cgacgtgaag cctcgctctc atttcc 26 31 28DNA Artificial Sequence Description of Artificial Sequence Primer 31cgatctcctt caagggaacc tagtgctg 28 32 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 32 gagttcacgc atgtgcaccg atgct25 33 25 DNA Artificial Sequence Description of Artificial SequencePrimer 33 cgtagcgtct tccaggatgt cctac 25 34 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 34 gcttggcctc gtctactccc gcaac25

We claim:
 1. A transgenic plant cell transformed with a nucleic acidencoding a polypeptide, wherein the polypeptide is defined in SEQ IDNO:8.
 2. The transgenic plant cell of claim 1, wherein the nucleic acidcomprises a polynucleotide as defined in SEQ ID NO:5.
 3. A transgenicplant cell transformed with a nucleic acid encoding a polypeptide,wherein expression of the polypeptide in the plant cell results in theplant cell's increased tolerance to an environmental stress selectedfrom one or more of the group consisting of drought and temperature lessthan or equal to 0° C., as compared to a wild type variety of the plantcell; wherein the nucleic acid hybridizes under stringent conditions toat least one sequence from the group consisting of a sequence of SEQ IDNO:5 and the full-length complement of the sequence of SEQ ID NO:5; andwherein the stringent conditions comprise hybridization in a 6× sodiumchloride/sodium citrate (SSC) solution at 65° C. and at least one washin a 0.2×SSC, 0.1% SDS solution at 50° C.
 4. A transgenic plant celltransformed with a nucleic acid encoding a polypeptide having at least90% sequence identity with a polypeptide as defined in SEQ ID NO:8,wherein expression of the polypeptide in the plant cell results in theplant cell's increased tolerance to an environmental stress selectedfrom one or more of the group consisting of drought and temperature lessthan or equal to 0° C., as compared to a wild type variety of the plantcell.
 5. The transgenic plant cell of any of claims 1, 2, 3, or 4,wherein the plant is a monocot.
 6. The transgenic plant cell of any ofclaims 1, 2, 3, or 4, wherein the plant is a dicot.
 7. The transgenicplant cell of any of claims 1, 2, 3, or 4, wherein the plant is selectedfrom the group consisting of maize, wheat, rye, oat, triticale, rice,barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper,sunflower, tagetes, solanaceous plants, potato, tobacco, eggplant,tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species,oil palm, coconut, and perennial grass.
 8. A transgenic plant comprisingthe transgenic plant cell according to any one of claims 1, 2, 3, or 4.9. A seed comprising the transgenic plant cell according to any one ofclaims 1, 2, 3, or
 4. 10. A seed produced by a transgenic plantcomprising a plant cell according to any of claims 1, 2, 3, or 4,wherein the seed comprises the nucleic acid encoding the polypeptide,wherein the seed is true breeding for an increased tolerance to anenvironmental stress as compared to a wild type variety of the plantcell, and wherein the environmental stress is selected from one or moreof the group consisting of drought and temperature less than or equal to0° C.
 11. An isolated nucleic acid encoding a polypeptide, wherein thenucleic acid comprises a polynucleotide that encodes the polypeptide asdefined in SEQ ID NO:8.
 12. The nucleic acid of claim 11, wherein thenucleic acid comprises the polynucleotide as defined in SEQ ID NO:5. 13.An isolated nucleic acid encoding a polypeptide, wherein expression ofthe polypeptide in the plant cell results in the plant cell's increasedtolerance to an environmental stress selected from one or more of thegroup consisting of drought and temperature less than or equal to 0° C.,as compared to a wild type variety of the plant cell; wherein thenucleic acid hybridizes under stringent conditions to at least onesequence from the group consisting of a sequence of SEQ ID NO:5 and thefull-length complement of the sequence of SEQ ID NO:5; and wherein thestringent conditions comprise hybridization in a 6× sodiumchloride/sodium citrate (SSC) solution at 65° C. and at least one washin a 0.2×SSC, 0.1% SDS solution at 50° C.
 14. An isolated nucleic acidencoding a polypeptide having at least 90% sequence identity with apolypeptide as defined in SEQ ID NO:8, wherein expression of thepolypeptide in the plant cell results in the plant cell's increasedtolerance to an environmental stress selected from one or more of thegroup consisting of drought and temperature less than or equal to 0° C.,as compared to a wild type variety of the plant cell.
 15. A seedcomprising the isolated nucleic acid according to any one of claims 11,12, 13, or
 14. 16. An isolated recombinant expression vector comprisinga nucleic acid of any one of claims 11, 12, 13, or 14, whereinexpression of the polypeptide in a plant cell results in the plantcell's increased tolerance to an environmental stress as compared to awild type variety of the plant cell, and wherein the environmentalstress is selected from one or more of the group consisting of droughtand temperature less than or equal to 0° C.
 17. A method of producing atransgenic plant comprising a nucleic acid encoding a polypeptide,comprising, a. transforming a plant cell with the expression vector ofclaim 16; and b. generating from the plant cell a transgenic plant thatexpresses the polypeptide; wherein the polypeptide is defined in SEQ IDNO:8.
 18. The method of claim 17, wherein the expression vectorcomprises the polynucleotide as defined in SEQ ID NO:5.
 19. A method ofproducing a transgenic plant comprising a nucleic acid encoding apolypeptide, wherein expression of the polypeptide in the plant resultsin the plant's increased tolerance to an environmental stress ascompared to a wild type variety of the plant, comprising, a.transforming a plant cell with the expression vector of claim 16; and b.generating from the plant cell a transgenic plant that expresses thepolypeptide; wherein the nucleic acid hybridizes under stringentconditions to at least one sequence from the group consisting of asequence of SEQ ID NO:5 and the full-length complement of the sequenceof SEQ ID NO:5; wherein the stringent conditions comprise hybridizationin a 6× sodium chloride/sodium citrate (SSC) solution at 65° C. and atleast one wash in a 0.2×SSC, 0.1% SDS solution at 50° C.; and whereinthe environmental stress is selected from one or more of the groupconsisting of drought and temperature less than or equal to 0° C.
 20. Amethod of producing a transgenic plant comprising a nucleic acidencoding a polypeptide, wherein expression of the polypeptide in theplant results in the plant's increased tolerance to an environmentalstress as compared to a wild type variety of the plant, comprising, a.transforming a plant cell with the expression vector of claim 16; and b.generating from the plant cell a transgenic plant that expresses thepolypeptide; wherein the polypeptide has at least 90% sequence identitywith the polypeptide as defined in SEQ ID NO:5, and wherein theenvironmental stress is selected from one or more of the groupconsisting of drought and temperature less than or equal to 0° C.